Glial mitogenic factors

ABSTRACT

The invention is concerned with new glial growth factors, such as the molecule referred to as GGF2. These factors are mitogenic factors for, e.g., Schwann cells.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 08/036,555filed Mar. 24, 1993, which is a continuation-in-part of Ser. No.07/863,703 filed Apr. 3, 1992 (abandoned), which in turn is acontinuation-in-part of Ser. No. 07/907,138 filed Jun. 30, 1992(abandoned), which is a continuation-in-part of Ser. No. 07/940,389filed Sep. 3, 1992 (abandoned), which is a continuation-in-part of Ser.No. 07/965,173 filed Oct. 23, 1992 (abandoned).

BACKGROUND OF THE INVENTION

This invention relates to polypeptides found in vertebrate species,which polypeptides are mitogenic growth factors for glial cells,including Schwann cells. The invention is also concerned with processescapable of producing such factors, and the therapeutic application ofsuch factors.

The glial cells of vertebrates constitute the specialized connectivetissue of the central and peripheral nervous systems. Important glialcells include Schwann cells which provide metabolic support for neuronsand which provide myelin sheathing around the axons of certainperipheral neurons, thereby forming individual nerve fibers. Schwanncells support neurons and provide a sheath effect by forming concentriclayers of membrane around adjacent neural axons, twisting as theydevelop around the axons. These myelin sheaths are a susceptible elementof many nerve fibers, and damage to Schwann cells, or failure in growthand development, can be associated with significant demyelination ornerve degeneration characteristic of a number of peripheral nervoussystem diseases and disorders. In the development of the nervous system,it has become apparent that cells require various factors to regulatetheir division and growth, and various such factors have been identifiedin recent years, including some found to have an effect on Schwann celldivision or development.

Thus, Brockes et al., inter alia, in J. Neuroscience, 4 (1984) 75-83describe a protein growth factor present in extracts from bovine brainand pituitary tissue, which was named Glial Growth Factor (GGF). Thisfactor stimulated cultured rat Schwann cells to divide against abackground medium containing ten percent fetal calf serum. The factorwas also described as having a molecular weight of 31,000 Daltons and asreadily dimerizing. In Meth. Enz., 147 (1987), 217-225, Brockesdescribes a Schwann cell-based assay for GGF.

Brockes et al., supra, also describes a method of purification of GGF toapparent homogeneity. In brief, one large-scale purification methoddescribed involves extraction of the lyophilized bovine anterior lobesand chromatography of material obtained thereby using NaCl gradientelution from CM cellulose. Gel filtration is then carried out with anUltrogel column, followed by elution from a phosphocellulose column, andfinally, small-scale SDS gel electrophoresis. Alternatively, theCM-cellulose material was applied directly to a phosphocellulose column,fractions from the column were pooled and purified by preparative nativegel electrophoresis, followed by a final SDS gel electrophoresis.

Brockes et al. observe that in previously reported gel filtrationexperiments (Brockes et al., J. Biol. Chem. 255 (1980) 8374-8377), themajor peak of growth factor activity was observed to migrate with amolecular weight of 56,000 Daltons, whereas in the first of theabove-described procedures activity was predominantly observed atmolecular weight 31,000. It is reported that the GGF dimer is largelyremoved as a result of the gradient elution from CM-cellulose in thisprocedure.

Benveniste et al. (PNAS, 82 (1985), 3930-3934) describe a Tlymphocyte-derived glial growth promoting factor. This factor, underreducing conditions, exhibits a change in apparent molecular weight onSDS gels.

Kimura et al. (Nature, 348 (1990), 257-260) describe a factor they termSchwannoma-derived growth factor which is obtained from a sciatic nervesheath tumor. The authors state that SDGF does not stimulate theincorporation of tritium-labelled TdR into cultured Schwann cells underconditions where, in contrast, partially purified pituitary fractioncontaining GGF is active. SDGF has an apparent molecular weight ofbetween 31,000 and 35,000.

Davis and Stroobant (J. Cell. Biol., 110 (1990), 1353-1360) describe thescreening of a number of candidate mitogens. Rat Schwann cells wereused, the chosen candidate substances being examined for their abilityto stimulate DNA synthesis in the Schwann cells in the presence of 10%FCS (fetal calf serum), with and without forskolin. One of the factorstested was GGF-carboxymethyl cellulose fraction (GGF-CM), which wasmitogenic in the presence of FCS, with and without forskolin. The workrevealed that in the presence of forskolin, inter alia, platelet derivedgrowth factor (PDGF) was a potent mitogen for Schwann cells, PDGF havingpreviously been thought to have no effect on Schwann cells.

Holmes et al. Science (1992) 256: 205 and Wen et al. Cell (1992) 69: 559demonstrate that DNA sequences which encode proteins binding to areceptor (p185^(erbB2)) are associated with several human tumors.

The p185^(erbB2) protein is a 185 kilodalton membrane spanning proteinwith tyrosine kinase activity. The protein is encoded by the erbB2proto-oncogene (Yarden and Ullrich Ann. Rev. Biochem. 57: 443 (1988)).The erbB2 gene, also referred to as HER-2 (in human cells) and neu (inrat cells), is closely related to the receptor for epidermal growthfactor (EGF). Recent evidence indicates that proteins which interactwith (and activate the kinase of) p185^(erbB2) induce proliferation inthe cells bearing p185^(erbB2) (Holmes et al. Science 256: 1205 (1992);Dobashi et al. Proc. Natl. Acad. Sci. 88: 8582 (1991); Lupu et al. Proc.Natl. Acad. Sci. 89: 2287 (1992)). Furthermore, it is evident that thegene encoding p185^(erbB2) binding proteins produces a number ofvariably-sized, differentially-spliced RNA transcripts that give rise toa series of proteins, which are of different lengths and contain somecommon peptide sequences and some unique peptide sequences. This issupported by the differentially-spliced RNA transcripts recoverable fromhuman breast cancer (MDA-MB-231) (Holmes et al. Science 256: 1205(1992)). Further support derives from the wide size range of proteinswhich act as (as disclosed herein) ligands for the p185^(erbB2) receptor(see below).

SUMMARY OF THE INVENTION

In general the invention provides methods for stimulating glial cell (inparticular, Schwann cell and glia of the central nervous system)mitogenesis, as well as new proteins exhibiting such glial cellmitogenic activity. In addition, DNA encoding these proteins andantibodies which bind these and related proteins are provided.

The novel proteins of the invention include alternative splicingproducts of sequences encoding known polypeptides. Generally, theseknown proteins are members of the GGF/p185^(erbB2) family of proteins.

Specifically, the invention provides polypeptides of a specifiedformula, and DNA sequences encoding those polypeptides. The polypeptideshave the formula

    WYBAZCX

wherein WYBAZCX is composed of the amino acid sequences shown in FIG. 31(SEQ ID Nos. 136-139, 141-147, 160, 161); wherein W comprises thepolypeptide segment F, or is absent; wherein Y comprises the polypeptidesegment E, or is absent; wherein Z comprises the polypeptide segment Gor is absent; and wherein X comprises the polypeptide segments C/D HKL,C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL,C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL,C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, orC/D C/D' D' HKL; provided that, either

a ) at least one of F, Y, B, A, Z, C, or X is of bovine origin; or

b) Y comprises the polypeptide segment E; or

c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKL, C/DC/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D'D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, C/D'H, C/DC/D'H, or C/D C/D' HL.

In addition, the invention includes the DNA sequence comprising codingsegments ^(5') FBA^(3') as well as the with corresponding polypeptidesegments having the amino acid sequences shown in FIG. 31 (SEQ ID Nos.136, 138, 139);

the DNA sequence comprising the coding segments ^(5') FBA^(3') as wellas the corresponding polypeptide segments having the amino acidsequences shown in FIG. 31 (SEQ ID Nos. 136, 138, 140);

the DNA sequence comprising the coding segments ^(5') FEBA^(3') as wellas the corresponding polypeptide segments having the amino acidsequences shown in FIG. 31 (SEQ ID Nos. 136-139);

the DNA sequence comprising the coding segments ^(5') FEBA'^(3') as wellas the corresponding polypeptide segments having the amino acidsequences shown in FIG. 31 (SEQ ID Nos. 136-138, 140); and

the DNA sequence comprising the polypeptide coding segments of theGGF2HBS5 cDNA clone (ATCC Deposit No. 75298, deposited Sep. 2, 1992).

The invention further includes peptides of the formula FBA, FEBA, FBA'FEBA' and DNA sequences encoding these peptides wherein the polypeptidesegments correspond to amino acid sequences shown in FIG. 31, SEQ IDNos. (136, 138 and 139), (136-139) and (136, 138 and 140 and (136-138and 140) respectively. The purified GGF-II polypeptide (SEQ ID No. 167)is also included as a part of the invention.

Further included as an aspect of the invention are peptides and DNAencoding such peptides which are useful for the treatment of glia and inparticular oligodendrocytes, microglia and astrocytes, of the centralnervous system and methods for the administration of these peptides.

The invention further includes vectors including DNA sequences whichencode the amino acid sequences, as defined above. Also included are ahost cell containing the isolated DNA encoding the amino acid sequences,as defined above. The invention further includes those compounds whichbind the p185^(erbB2) receptor and stimulate glial cell mitogenesis invivo and/or in vitro.

Also a part of the invention are antibodies to the novel peptidesdescribed herein. In addition, antibodies to any of the peptidesdescribed herein may be used for the purification of polypeptidesdescribed herein. The antibodies to the polypeptides may also be usedfor the therapeutic inhibition of glial cell mitogenesis.

The invention further provides a method for stimulating glial cellmitogenesis comprising contacting glial cells with a polypeptide definedby the formula

    WYBAZCX

wherein WYBAZCX is composed of the polypeptide segments shown in FIG. 31(SEQ ID Nos. 136-139, 141-147, 160, 161); wherein W comprises thepolypeptide segment F, or is absent wherein Y comprises the polypeptidesegment E, or is absent; wherein Z comprises the polypeptide segment Gor is absent; and wherein X comprises the polypeptide segment C/D HKL,C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL,C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL,C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, orC/D C/D' D' HKL.

The invention also includes a method for the preparation of a glial cellmitogenic factor which consist of culturing modified host cells asdefined above under conditions permitting expression of the DNAsequences of the invention.

The peptides of the invention can be used to make a pharmaceutical orveterinary formulation for pharmaceutical or veterinary use. Optionally,the formulation may be used together with an acceptable diluent, carrieror excipient and/or in unit dosage form.

A method for stimulating mitogenesis of a glial cell by contacting theglial cell with a polypeptide defined above as a glial cell mitogen invivo or in vitro is also an aspect of the invention. A method forproducing a glial cell mitogenic effect in a vertebrate (preferably amammal, more preferably a human) by administering an effective amount ofa polypeptide as defined is also a component of the invention.

Methods for treatment of diseases and disorders using the polypeptidesdescribed are also a part of the invention. For instance, a method oftreatment or prophylaxis for a nervous disease or disorder can beeffected with the polypeptides described. Also included are a method forthe prophylaxis or treatment of a pathophysiological condition of thenervous system in which a cell type is involved which is sensitive orresponsive to a polypeptide as defined are a part of the invention.

Included in the invention as well, are methods for treatment when thecondition involves peripheral nerve damage; nerve damage in the centralnervous system; neurodegenerative disorders; demyelination in peripheralor central nervous system; or damage or loss of Schwann cellsoligodendrocytes, microgila, or astrocytes. For example a neuropathy ofsensory or motor nerve fibers; or the treatment of a neurodegenerativedisorder are included. In any of these cases, treatment consists ofadministering an effective amount of the polypeptide.

The invention also includes a method for inducing neural regenerationand/or repair by administering an effective amount of a polypeptide asdefined above. Such a medicament is made by administering thepolypeptide with a pharmaceutically effective carrier.

The invention includes the use of a polypeptide as defined above in themanufacture of a medicament.

The invention further includes the use of a polypeptide as defined above

to immunize a mammal for producing antibodies, which can optionally beused for therapeutic or diagnostic purposes

in a competitive assay to identify or quantify molecules having receptorbinding characteristics corresponding to those of the polypeptide;and/or

for contacting a sample with a polypeptide, as mentioned above, alongwith a receptor capable of binding specifically to the polypeptide forthe purpose of detecting competitive inhibition of binding to thepolypeptide.

in an affinity isolation process, optionally affinity chromatography,for the separation of a corresponding receptor.

The invention also includes a method for the prophylaxis or treatment ofa glial tumor. This method consists of administering an effective amountof a substance which inhibits the binding of a factor as defined by thepeptides above.

Furthermore, the invention includes a method of stimulating glial cellmitogenic activity by the application to the glial cell of a

30 kD polypeptide factor isolated from the MDA--MB 231 human breast cellline; or

35 kD polypeptide factor isolated from the rat I-EJ transformedfibroblast cell line to the glial cell or

75 kD polypeptide factor isolated from the SKBR-3 human breast cellline; or

44 kD polypeptide factor isolated from the rat I-EJ transformedfibroblast cell line; or

25 kD polypeptide factor isolated from activated mouse peritonealmacrophages; or

45 kD polypeptide factor isolated from the MDA--MB 231 human breastcell; or

7 to 14 kD polypeptide factor isolated from the ATL-2 human T-cell lineto the glial cell; or

25 kD polypeptide factor isolated from the bovine kidney cells; or

42 kD polypeptide factor (ARIA) isolated from brains.

The invention further includes a method for the use of the EGFL1, EGFL2,EGFL3, EGFL4, EGFL5, and EGFL6 polypeptides, FIGS. 38 to 43 and SEQ IDNos. 154 to 159, respectively, for the stimulation of glial cellmitogenesis in vivo and in vitro.

Also included in the invention is the administration of the GGF-IIpolypeptide whose sequence is shown in FIG. 45 for the stimulation ofglial cell mitogenesis.

An additional aspect of the invention includes the use of theabove-referenced peptides for the purpose of stimulating Schwann cellsto produce growth factors which may, in turn, be harvested forscientific or therapeutic use.

Furthermore, the peptides described herein may be used to induce centralglial proliferation and remyelination for treatment of diseases, e.g.,MS, where re-myelination is desired.

In an additional aspect of the invention, the novel polypeptidesdescribed herein may be used to stimulate the synthesis of acetylcholinereceptors.

As mentioned above, the invention provides new glial growth factors frommammalian sources, including bovine and human, which are distinguishedfrom known factors. These factors are mitogenic for Schwann cellsagainst a background of fetal calf plasma (FCP). The invention alsoprovides processes for the preparation of these factors, and an improvedmethod for defining activity of these and other factors. Therapeuticapplication of the factors is a further significant aspect of theinvention.

Thus, important aspects of the invention are:

(a) a basic polypeptide factor having glial cell mitogenic activity,more specifically, Schwann cell mitogenic activity in the presence offetal calf plasma, a molecular weight of from about 30 kD to about 36kD, and including within its amino acid sequence any one or more of thefollowing peptide sequences: ##STR1## (b) a basic polypeptide factorwhich stimulates glial cell mitogenesis, particularly the division ofSchwann cells, in the presence of fetal calf plasma, has a molecularweight of from about 55 kD to about 63 kD, and including within itsamino acid sequence any one or more of the following peptide sequences:##STR2##

The novel peptide sequences set out above, derived from the smallermolecular weight polypeptide factor, and from the larger molecularweight polypeptide factor, are also aspects of this invention in theirown right. These sequences are useful as probe sources for polypeptidefactors of the invention, for investigating, isolating or preparing suchfactors (or corresponding gene sequences) from a range of differentspecies, or preparing such factors by recombinant technology, and in thegeneration of corresponding antibodies, by conventional technologies,preferably monoclonal antibodies, which are themselves usefulinvestigative tools and are possible therapeutics. The invention alsoincludes an isolated glial cell mitogenic activity encoding genesequence, or fragment thereof, obtainable by the methods set out abovefor the novel peptide sequences of the invention.

The availability of short peptides from the highly purified factors ofthe invention has enabled additional sequences to be determined (seeExamples to follow).

Thus, the invention further embraces a polypeptide factor having glialcell mitogenic activity and including an amino acid sequence encoded by:

(a) a DNA sequence shown in any one of FIGS. 28a, 28b or 28c, SEQ IDNos. 133-135, respectively;

(b) a DNA sequence shown in FIG. 22, SEQ ID No. 89;

(c) the DNA sequence represented by nucleotides 281-557 of the sequenceshown in FIG. 28a, SEQ ID No. 133; or

(d) a DNA sequence hybridizable to any one of the DNA sequencesaccording to (a), (b) or (c).

The invention further includes sequences which have greater than 60%,preferably 80%, sequence identity of homology to the sequences indicatedabove.

While the present invention is not limited to a particular set ofhybridization conditions, the following protocol gives general guidancewhich may, if desired, be followed:

DNA probes may be labelled to high specific activity (approximately 10⁸to 10⁹ 32 Pdmp/μg) by nick-translation or by PCR reactions according toSchowalter and Sommer (Anal. Biochem., 177:90-94, 1989) and purified bydesalting on G-150 Sephadex columns. Probes may be denatured (10 minutesin boiling water followed by immersion into ice water), then added tohybridization solutions of 80% buffer B (2 g polyvinylpyrolidine, 2 gFicoll-400, 2 g bovine serum albumin, 50 ml 1M Tris HCL (pH 7.5), 58 gNaCl, 1 g sodium pyrophosphate, 10 g sodium dodecyl sulfate, 950 ml H₂O) containing 10% dextran sulfate at 10⁶ dpm ³² P per ml and incubatedovernight (approximately 16 hours) at 60° C. The filters may then bewashed at 60° C., first in buffer B for 15 minutes followed by three20-minute washes in 2× SSC, 0.1% SDS then one for 20 minutes in 1× SSC,0.1% SDS.

In other respects, the invention provides:

(a) a basic polypeptide factor which has, if obtained from bovinepituitary material, an observed molecular weight, whether in reducingconditions or not, of from about 30 kD to about 36 kD onSDS-polyacrylamide gel electrophoresis using the following molecularweight standards:

    ______________________________________                                        Lysozyme (hen egg white) 14,400                                               Soybean trypsin inhibitor                                                                              21,500                                               Carbonic anhydrase (bovine)                                                                            31,000                                               Ovalbumin (hen egg white)                                                                              45,000                                               Bovine serum albumin     66,200                                               Phosphorylase B (rabbit muscle)                                                                        97,400;                                              ______________________________________                                    

which factor has glial cell mitogenic activity including stimulating thedivision of rat Schwann cells in the presence of fetal calf plasma, andwhen isolated using reversed-phase HPLC retains at least 50% of saidactivity after 10 weeks incubation in 0.1% trifluoroacetic acid at 4°C.; and

(b) a basic polypeptide factor which has, if obtained from bovinepituitary material, an observed molecular weight, under non-reducingconditions, of from about 55 kD to about 63 kD on SDS-polyacrylamide gelelectrophoresis using the following molecular weight standards:

    ______________________________________                                        Lysozyme (hen egg white) 14,400                                               Soybean trypsin inhibitor                                                                              21,500                                               Carbonic anhydrase (bovine)                                                                            31,000                                               Ovalbumin (hen egg white)                                                                              45,000                                               Bovine serum albumin     66,200                                               Phosphorylase B (rabbit muscle)                                                                        97,400;                                              ______________________________________                                    

which factor the human equivalent of which is encoded by DNA cloneGGF2HBS5 described herein and which factor has glial cell mitogenicactivity including stimulating the division of rat Schwann cells in thepresence of fetal calf plasma, and when isolated using reversed-phaseHPLC retains at least 50% of the activity after 4 days incubation in0.1% trifluoroacetic acid at 4° C.

For convenience of description only, the lower molecular weight andhigher molecular weight factors of this invention are referred tohereafter as "GGF-I" and "GGF-II", respectively. The "GGF2" designationis used for all clones isolated with peptide sequence data derived fromGGF-II protein (i.e., GGF2HBS5, GGF2BPP3).

It will be appreciated that the molecular weight range limits quoted arenot exact, but are subject to slight variations depending upon thesource of the particular polypeptide factor. A variation of, say, about10% would not, for example, be impossible for material from anothersource.

Another important aspect of the invention is a DNA sequence encoding apolypeptide having glial cell mitogenic activity and comprising:

(a) a DNA sequence shown in any one of FIGS. 28a, 28b or 28c, SEQ IDNos. 133-135:

(b) a DNA sequence shown in FIG. 22, SEQ ID No. 89;

(c) the DNA sequence represented by nucleotides 281-557 of the sequenceshown in FIG. 28a, SEQ ID No. 133; or

(d) a DNA sequence hybridizable to any one of the DNA sequencesaccording to (a), (b) or (c).

Another aspect of the present invention uses the fact that the GlialGrowth Factors and p185^(erbB2) ligand proteins are encoded by the samegene. A variety of messenger RNA splicing variants (and their resultantproteins) are derived from this gene and many of these products showp185^(erbB2) binding and activation. Several of the (GGF-II) geneproducts have been used to show Schwann cell mitogenic activity. Thisinvention provides a use for all of the known products of theGGF/p185^(erbB2) ligand gene (described in the references listed above)as Schwann cell mitogens.

This invention also relates to other, not yet naturally isolatedsplicing variants of the Glial Growth Factor gene. FIG. 30, shows theknown patterns of splicing derived from polymerase chain reactionexperiments (on reverse transcribed RNA) and analysis of cDNA clones (aspresented within) and derived from what has been published as sequencesencoding p185^(erbB2) ligands (Peles et al., Cell 69: 205 (1992) and Wenet al., Cell 69: 559 (1992)). These patterns, as well as additional onesdisclosed herein, represent probable splicing variants which exist. Thusanother aspect of the present invention relates to the nucleotidesequences encoding novel protein factors derived from this gene. Theinvention also provides processes for the preparation of these factors.Therapeutic application of these new factors is a further aspect of theinvention.

Thus other important aspects of the invention are:

(a) A series of human and bovine polypeptide factors having glial cellmitogenic activity including stimulating the division of Schwann cells.These peptide sequences are shown in FIGS. 31, 32, 33 and 34, SEQ IDNos. 136-137, respectively.

(b) A series of polypeptide factors having glial cell mitogenic activityincluding stimulating the division of Schwann cells and purified andcharacterized according to the procedures outlined by Lupu et al.Science 249: 1552 (1990); Lupu et al. Proc. Natl. Acad. Sci USA 89: 2287(1992); Holmes et al. Science 256: 1205 (1992); Peles et al. 69: 205(1992); Yarden and Pales Biochemistry 30: 3543 (1991); Dobashi et al.Proc. Natl. Acad. Sci. 88: 8582 (1991); Davis et al. Biochem. Biophys.Res. Commun. 179: 1536 (1991); Beaumont et al., patent applicationPCT/US91/03443 (1990); Greene et al. patent application PCT/US91/02331(1990); Usdin and Fischbach, J. Cell. Biol. 103: 493-507 (1986); Fallset al., Cold Spring Harbor Symp. Quant. Biol. 55: 397-406 (1990); Harriset al., Proc. Natl. Acad. Sci. USA 88: 7664-7668 (1991); and Falls etal., Cell 72: 801-815 (1993).

(c) A polypeptide factor (GGFBPP5) having glial cell mitogenic activityincluding stimulating the division of Schwann cells. The amino acidsequence is shown in FIG. 32, SEQ ID No. 148, and is encoded by thebovine DNA sequence shown in FIG. 32, SEQ ID No. 148.

The novel human peptide sequences described above and presented in FIGS.31, 32, 33 and 34, SEQ ID Nos. 136-150, respectively, represent a seriesof splicing variants which can be isolated as full length complementaryDNAs (cDNAs) from natural sources (cDNA libraries prepared from theappropriate tissues) or can be assembled as DNA constructs withindividual exons (e.g., derived as separate exons) by someone skilled inthe art.

Other compounds in particular, peptides, which bind specifically to thep185^(erbB2) receptor can also be used according to the invention as aglial cell mitogen. A candidate compound can be routinely screened forp185^(erbB2) binding, and, if it binds, can then be screened for glialcell mitogenic activity using the methods described herein.

The invention includes any modifications or equivalents of the abovepolypeptide factors which do not exhibit a significantly reducedactivity. For example, modifications in which amino acid content orsequence is altered without substantially adversely affecting activityare included. By way of illustration, in EP-A 109748 mutations of nativeproteins are disclosed in which the possibility of unwanted disulfidebonding is avoided by replacing any cysteine in the native sequencewhich is not necessary for biological activity with a neutral aminoacid. The statements of effect and use contained herein are therefore tobe construed accordingly, with such uses and effects employing modifiedor equivalent factors being part of the invention.

The new sequences of the invention open up the benefits of recombinanttechnology. The invention thus also includes the following aspects:

(a) DNA constructs comprising DNA sequences as defined above in operablereading frame position within vectors (positioned relative to controlsequences so as to permit expression of the sequences) in chosen hostcells after transformation thereof by the constructs (preferably thecontrol sequence includes regulatable promoters, e.g. Trp). It will beappreciated that the selection of a promoter and regulatory sequences(if any) are matters of choice for those of skill in the art;

(b) host cells modified by incorporating constructs as defined in (a)immediately above so that said DNA sequences may be expressed in saidhost cells--the choice of host is not critical, and chosen cells may beprokaryotic or eukaryotic and may be genetically modified to incorporatesaid constructs by methods known in the art; and,

(c) a process for the preparation of factors as defined above comprisingcultivating the modified host cells under conditions permittingexpression of the DNA sequences. These conditions can be readilydetermined, for any particular embodiment, by those of skill in the artof recombinant DNA technology. Glial cell mitogens prepared by thismeans are included in the present invention.

None of the factors described in the art has the combination ofcharacteristics possessed by the present new polypeptide factors.

As indicated, the Schwann cell assay used to characterize the presentfactors employs a background of fetal calf plasma. In all otherrespects, the assay can be the same as that described by Brockes et al.in Meth. Enz., supra, but with 10% FCP replacing 10% FCS. Thisdifference in assay techniques is significant, since the absence ofplatelet-derived factors in fetal calf plasma (as opposed to serum)enables a more rigorous definition of activity on Schwann cells byeliminating potentially spurious effects from some other factors.

The invention also includes a process for the preparation of apolypeptide as defined above, extracting vertebrate brain material toobtain protein, subjecting the resulting extract to chromatographicpurification by hydroxylapatite HPLC and then subjecting these fractionsto SDS-polyacrylamide gel electrophoresis. The fraction which has anobserved molecular weight of about 30 kD to 36 kD and/or the fractionwhich has an observed molecular weight of about 55 kD to 63 kD iscollected. In either case, the fraction is subjected toSDS-polyacrylamide gel electrophoresis using the following molecularweight standards:

    ______________________________________                                        Lysozyme (hen egg white) 14,400                                               Soybean trypsin inhibitor                                                                              21,500                                               Carbonic anhydrase (bovine)                                                                            31,000                                               Ovalbumin (hen egg white)                                                                              45,000                                               Bovine serum albumin     66,200                                               Phosphorylase B (rabbit muscle)                                                                        97,400                                               ______________________________________                                    

In the case of the smaller molecular weight fraction, theSDS-polyacrylamide gel is run in non-reducing conditions in reducingconditions or, and in the case of the larger molecular weight fractionthe gel is run under non-reducing conditions. The fractions are thentested for activity stimulating the division of rat Schwann cellsagainst a background of fetal calf plasma.

Preferably, the above process starts by isolating a relevant fractionobtained by carboxymethyl cellulose chromatography, e.g. from bovinepituitary material. It is also preferred that hydroxylapatite HPLC,cation exchange chromatography, gel filtration, and/or reversed-phaseHPLC be employed prior to the SDS-Polyacrylamide gel electrophoresis. Ateach stage in the process, activity may be determined using Schwann cellincorporation of radioactive iododeoxyuridine as a measure in an assaygenerally as described by Brockes in Meth. Enz., supra, but modified bysubstituting 10% FCP for 10% FCS. As already noted, such as assay is anaspect of the invention in its own substance for CNS or PNS cell, e.g.Schwann cell, mitogenic effects.

Thus, the invention also includes an assay for glial cell mitogenicactivity in which a background of fetal calf plasma is employed againstwhich to assess DNA synthesis in glial cells stimulated (if at all) by asubstance under assay.

Another aspect of the invention is a pharmaceutical or veterinaryformulation comprising any factor as defined above formulated forpharmaceutical or veterinary use, respectively, optionally together withan acceptable diluent, carrier or excipient and/or in unit dosage form.In using the factors of the invention, conventional pharmaceutical orveterinary practice may be employed to provide suitable formulations orcompositions.

Thus, the formulations of this invention can be applied to parenteraladministration, for example, intravenous, subcutaneous, intramuscular,intraorbital, opthalmic, intraventricular, intracranial, intracapsular,intraspinal, intracisternal, intraperitoneal, topical, intranasal,aerosol, scarification, and also oral, buccal, rectal or vaginaladministration.

The formulations of this invention may also be administered by thetransplantation into the patient of host cells expressing the DNA of theinstant invention or by the use of surgical implants which release theformulations of the invention.

Parenteral formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are to be foundin, for example, "Remington's Pharmaceutical Sciences." Formulations forparenteral administration may, for example, contain as excipientssterile water or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, or hydrogenated naphthalenes,biocompatible, biodegradable lactide polymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the present factors. Other potentially useful parenteraldelivery systems for the factors include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain as excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel to be applied intranasally. Formulations for parenteraladministration may also include glycocholate for buccal administration,methoxysalicylate for rectal administration, or citric acid for vaginaladministration.

The present factors can be used as the sole active agents, or can beused in combination with other active ingredients, e.g., other growthfactors which could facilitate neuronal survival in neurologicaldiseases, or peptidase or protease inhibitors.

The concentration of the present factors in the formulations of theinvention will vary depending upon a number of issues, including thedosage to be administered, and the route of administration.

In general terms, the factors of this invention may be provided in anaqueous physiological buffer solution containing about 0.1 to 10% w/vcompound for parenteral administration. General dose ranges are fromabout 1 mg/kg to about 1 g/kg of body weight per day; a preferred doserange is from about 0.01 mg/kg to 100 mg/kg of body weight per day. Thepreferred dosage to be administered is likely to depend upon the typeand extent of progression of the pathophysiological condition beingaddressed, the overall health of the patient, the make up of theformulation, and the route of administration.

As indicated above, Schwann cells (the glial cells of the peripheralnervous system) are stimulated to divide in the presence of the factorsof the invention. Schwann cells of the peripheral nervous system areinvolved in supporting neurons and in creating the myelin sheath aroundindividual nerve fibers. This sheath is important for proper conductionof electrical impulses to muscles and from sensory receptors.

There are a variety of peripheral neuropathies which Schwann cells andnerve fibers are damaged, either primarily or secondarily. There aremany neuropathies of both sensory and motor fibers (Adams and Victor,Principles of Neurology). The most important of those neuropathies areprobably the neuropathies associates with diabetes, multiple sclerosis,Landry-Guillain-Barr syndrome, neuropathies caused by carcinomas, andneuropathies caused by toxic agents (some of which are used to treatcarcinomas).

The invention, however, envisages treatment or prophylaxis of conditionswhere nervous system damage has been brought about by any basic cause,e.g. infection or injury. Thus, in addition to use of the presentfactors in the treatment of disorders or diseases of the nervous systemwhere demyelination or loss of Schwann cells is present, such glialgrowth factors can be valuable in the treatment of disorders of thenervous system that have been caused by damage to the peripheral nerves.Following damage to peripheral nerves, the regeneration process is ledby the growth or the re-establishment of Schwann cells, followed by theadvancement of the nerve fibre back to its target. By speeding up thedivision of Schwann cells one could promote the regenerative processfollowing damage.

Similar approaches could be used to treat injuries or neurodegenerativedisease of the central nervous system (brain and spinal cord).

Furthermore, there are a variety of tumors of glial cells the mostcommon of which is probably neurofibromatosis, which is a patchy smalltumor created by overgrowth of glial cells. Also, it has been found thatan activity very much like GGF can be found in some Schwann cell tumors,and therefore inhibitors of the action of the present factors on theirreceptors provides a therapy of a glial tumor, which comprisesadministering-an effective amount of a substance which inhibits thebinding of a factor, as defined above, to a receptor.

In general, the invention includes the use of present polypeptidefactors in the prophylaxis or treatment of any pathophysiologicalcondition of the nervous system in which a factor-sensitive orfactor-responsive cell type is involved.

The polypeptide factors of the invention can also be used as immunogensfor making antibodies, such as monoclonal antibodies, following standardtechniques. Such antibodies are included within the present invention.These antibodies can, in turn, be used for therapeutic or diagnosticpurposes. Thus, conditions perhaps associated with abnormal levels ofthe factor may be tracked by using such antibodies. In vitro techniquescan be used, employing assays on isolated samples using standardmethods. Imaging methods in which the antibodies are, for example,tagged with radioactive isotopes which can be imaged outside the bodyusing techniques for the art of tumour imaging may also be employed.

The invention also includes the general use of the present factors asglial cell mitogens in vivo or in vitro, and the factors for such use.One specific embodiment is thus a method for producing a glial cellmitogenic effect in a vertebrate by administering an effective amount ofa factor of the invention. A preferred embodiment is such a method inthe treatment or prophylaxis of a nervous system disease or disorder.

A further general aspect of the invention is the use of a factor of theinvention in the manufacture of a medicament, preferably for thetreatment of a nervous disease or disorder, or for neural regenerationor repair.

Also included in the invention are the use of the factors of theinvention in competitive assays to identify or quantify molecules havingreceptor binding characteristics corresponding to those of saidpolypeptides. The polypeptides may be labelled, optionally with aradioisotope. A competitive assay can identify both antagonists andagonists of the relevant receptor.

In another aspect, the invention provides the use of each one of thefactors of the invention in an affinity isolation process, optionallyaffinity chromatography, for the separation of a respectivecorresponding receptor. Such processes for the isolation of receptorscorresponding to particular proteins are known in the art, and a numberof techniques are available and can be applied to the factors of thepresent invention. For example, in relation to IL-6 and IFNγ the readeris referred to Novick, D.; et al., J. Chromatogr. (1990) 510: 331-7.With respect to gonadotropin releasing hormone reference is made toHazum, E., J. (1990) Chromatogr. 510: 233-8. In relation to G-CSFreference is made to Fukunaga, R., et al., J. Biol. Chem., 265:13386-90. In relation to IL-2 reference is made to Smart, J. E., et al.,(1990) J. Invest. Dermatol., 94: 158S-163S, and in relation to humanIFN-gamma reference is made to Stefanos, S, et al., (1989) J. InterferonRes., 9: 719-30.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings will first be described.

Drawings

FIGS. 1 to 8 relate to Example 1, and are briefly described below:

FIG. 1 is the profile for product from carboxymethylcellulosechromatography;

FIG. 2 is the profile for product from hydroxylapatite HPLC;

FIG. 3 is the profile for product from Mono S FPLC;

FIG. 4 is the profile for product from Gel filtration FPLC;

FIGS. 5 and 6 depict the profiles for the two partially purifiedpolypeptide products from reversed-phase HPLC; and

FIGS. 7 and 8 depict dose-response curves for the GGF-I and GGF-IIfractions from reversed-phase HPLC using either a fetal calf serum or afetal calf plasma background;

FIGS. 9 to 12 depict the peptide sequences derived from GGF-I andGGF-II, SEQ ID Nos. 1-20, 22-29, 32-53 and 169, (see Example 2hereinafter), FIGS. 10 and 12 specifically depict novel sequences:

In FIG. 10, Panel A, the sequences of GGF-I peptides used to designdegenerate oligonucleotide probes and degenerate PCR primers are listed(SEQ ID Nos. 20, '1, 22-29, and 17). Some of the sequences in Panel Awere also used to design synthetic peptides. Panel B is a listing of thesequences of novel peptides that were too short (less than 6 aminoacids) for the design of degenerate probes or degenerate PCR primers(SEQ ID Nos. 17 and 52);

In FIG. 11, various trypsin and lysyl endopeptidase C peptides derivedfrom GGF2 are present.

In FIG. 12, Panel A, is a listing of the sequences of GGF-II peptidesused to design degenerate oligonucleotide probes and degenerate PCRprimers (SEQ ID Nos. 45-52). Some of the sequences in Panel A were usedto design synthetic peptides. Panel B is a listing of the novel peptidethat was too short (less than 6 amino acids) for the design ofdegenerate probes or degenerate PCR primers (SEQ ID No. 53);

FIG. 13 compares two radio label incorporation assays, used indetermining DNA synthesis in Schwann cell cultures.

FIGS. 14A and 14B compare Br-UdR immuno reactivity and Br-UdR labelledcell number.

FIG. 15 shows the mitogenic response of rat sciatic nerve Schwann cellsto GGFs.

FIG. 16 shows DNA synthesis in rat sciatic nerve cells and 3T3fibroblasts in the presence of GGFs.

FIG. 17 depicts the mitogenic response of BHK 21 C13 cells to FCS andGGFs.

FIG. 18 presents data showing the survival and proliferation of BHK 21C13 cell micro cultures after 48 hours in the presence of GGFs.

FIG. 19: shows the mitogenic response of C6 cells to FCS.

FIG. 20: shows that the mitogenic response of C6 cells to a FGF andGGFs.

FIGS. 21 to 28 (a, b and c) relate to Example 4, below and are brieflydescribed below:

FIG. 21 is a listing of the degenerate oligonucleotide probes (SEQ IDNos. 54-88) designed from the novel peptide sequences in FIG. 10, PanelA and FIG. 12, Panel A;

FIG. 22 (SEQ ID No. 89) depicts a stretch of the putative bovine GGF-IIgene sequence from the recombinant bovine genomic phage GGF2BG1,containing the binding site of degenerate oligonucleotide probes 609 and650 (see FIG. 21, SEQ ID NOs. 69 and 72, respectively). The figure isthe coding strand of the DNA sequence and the deduced amino acidsequence in the third reading frame. The sequence of peptide 12 fromfactor 2 (bold) is part of a 66 amino acid open reading frame(nucleotides 75272);

FIG. 23 is the degenerate PCR primers (Panel A, SEQ IS Nos. 90-108) andunique PCR primers (Panel B, SEQ ID Nos. 109-119) used in experiments toisolate segments of the bovine GGF-II coding sequences present in RNAfrom posterior pituitary;

FIG. 24 depicts of the nine distinct contiguous bovine GGF-II cDNAstructures and sequences that were obtained in PCR amplificationexperiments using the list of primers in FIG. 7, Panels A and B, and RNAfrom posterior pituitary. The top line of the Figure is a schematic ofthe coding sequences which contribute to the cDNA structures that werecharacterized;

FIG. 25 is a physical map of bovine recombinant phage of GGF2BG1. Thebovine fragment is roughly 20 kb in length and contains two exons (bold)of the bovine GGF-II gene. Restriction sites for the enzymes Xbal, SpeI,Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map.Shaded portions correspond to fragments which were subcloned forsequencing;

FIG. 26 is a schematic of the structure of three alternative geneproducts of the putative bovine GGF-II gene. Exons are listed A throughE in the order of their discovery. The alternative splicing patterns 1,2 and 3 generate three overlapping deduced protein structures (GGF2BPP1,2, and 3), which are displayed in the various FIGS. 28a, b, c (describedbelow);

FIG. 27 (SEQ ID Nos. 120-132) is a comparison of the GGF-I and GGF-IIsequences identified in the deduced protein sequences shown in FIGS.28a, 28b and 28c (described below) with the novel peptide sequenceslisted in FIGS. 10 and 12. The Figure shows that six of the nine novelGGF-II peptide sequences are accounted for in these deduced proteinsequences. Two peptide sequences similar to GGF-I sequences are alsofound;

FIG. 28a (SEQ ID No. 133) is a listing of the coding strand DNA sequenceand deduced amino acid sequence of the cDNA obtained from splicingpattern number 1 in FIG. 26. This partial cDNA of the putative bovineGGF-II gene encodes a protein of 206 amino acids in length. Peptides inbold were those identified from the lists presented in FIGS. 10 and 12.Potential glycosylation sites are underlined (along with polyadenylationsignal AATAAA);

FIG. 28b (SEQ ID No. 134) is a listing of the coding strand DNA sequenceand deduced amino acid sequence of the cDNA obtained from splicingpattern number 2 in FIG. 26. This partial cDNA of the putative bovineGGF-II gene encodes a protein of 281 amino acids in length. Peptides inbold are those identified from the lists presented in FIGS. 10 and 12.Potential glycosylation sites are underlined (along with polyadenylationsignal AATAAA);

FIG. 28c (SEQ ID No. 135) is a listing of the coding strand DNA sequenceand deduced amino acid sequence of the cDNA obtained from splicingpattern number 3 in FIG. 26. This partial cDNA of the putative bovineGGF-II gene encodes a protein of 257 amino acids in length. Peptides inbold are those identified from the lists in FIGS. 10 and 12. Potentialglycosylation sites are underlined (along with polyadenylation signalAATAAA).

FIG. 29, which relates to Example 6 hereinafter, is an autoradiogram ofa cross hybridization analysis of putative bovine GGF-II gene sequencesto a variety of mammalian DNAs on a southern blot. The filter containslanes of EcoRI-digested DNA (5 μg per lane) from the species listed inthe Figure. The probe detects a single strong band in each DNA sample,including a four kilobase fragment in the bovine DNA as anticipated bythe physical map in FIG. 25. Bands of relatively minor intensity areobserved as well, which could represent related DNA sequences. Thestrong hybridizing band from each of the other mammalian DNA samplespresumably represents the GGF-II homologue of those species.

FIG. 30 is a diagram of representative splicing variants. The codingsegments are represented by F, E, B, A, G, C, C/D, C/D', D, D', H, K andL The location of the peptide sequences derived from purified proteinare indicated by "o".

FIG. 31 (SEQ ID Nos. 136-147, 160, 161) is a listing of the DNAsequences and predicted peptide sequences of the coding segments of GGF.Line 1 is a listing of the predicted amino acid sequences of bovine GGF,line 2 is a listing of the nucleotide sequences of bovine GGF, line 3 isa listing of the nucleotide sequences of human GGF (heregulin)(nucleotide base matches are indicated with a vertical line) and line 4is a listing of the predicted amino acid sequences of humanGGF/heregulin where it differs from the predicted bovine sequence.Coding segments E, A' and K represent only the bovine sequences. Codingsegment D' represents only the human (heregulin) sequence.

FIG. 32 (SEQ ID No. 148) is the predicted GGF2 amino acid sequence andnucleotide sequence of BPP5. The upper line is the nucleotide sequenceand the lower line is the predicted amino acid sequence.

FIG. 33 (SEQ ID No. 149) is the predicted amino acid sequence andnucleotide sequence of GGF2BPP2. The upper line is the nucleotidesequence and the lower line is the predicted amino acid sequence.

FIG. 34 (SEQ ID No. 150) is the predicted amino acid sequence andnucleotide sequence of GGF2BPP4. The upper line is the nucleotidesequence and the lower line is the predicted amino acid sequence.

FIG. 35 (SEQ ID Nos. 151-152) depicts the alignment of two GGF peptidesequences (GGF2bpp4 and GGF2bpp5) with the human EGF (hEGF). Asterisksindicate positions of conserved cysteines.

FIG. 36 depicts the level of GGF activity (Schwann cell mitogenic assay)and tyrosine phosphorylation of a ca. 200 kD protein (intensity of a 200kD band on an autoradiogram of a Western blot developed with anantiphosphotyrosine polyclonal antibody) in response to increasingamounts of GGF.

FIG. 37 is a list of splicing variants derived from the sequences shownin FIG. 31.

FIG. 38 is the predicted amino acid sequence, bottom, and nucleicsequence, top, of EGFL1 (SEQ ID No. 154).

FIG. 39 is the predicted amino acid sequence, bottom, and nucleicsequence, top, of EGFL2 (SEQ ID No. 155).

FIG. 40 is the predicted amino acid sequence, bottom, and nucleicsequence, top, of EGFL3 (SEQ ID No. 156).

FIG. 41 is the predicted amino acid sequence, bottom, and nucleicsequence, top, of EGFL4 (SEQ ID No. 157).

FIG. 42 is the predicted amino acid sequence, bottom, and nucleicsequence, top, of EGFL5 (SEQ ID No. 158).

FIG. 43 is the predicted amino acid sequence, bottom, and nucleicsequence, top, of EGFL6 (SEQ ID No. 159).

FIG. 44 is a scale coding segment map of the clone. T3 refers to thebacteriophage promoter used to produce mRNA from the clone. R=flankingEcoRI restriction enzyme sites. 5' UT refers to the 5' untranslatedregion. E, B, A, C, C/D', and D refer to the coding segments O=thetranslation start site. Λ=the 5' limit of the region homologous to thebovine E segment (see example 6) and 3' UT refers to the 3' untranslatedregion.

FIG. 45 is the predicted amino acid sequence (middle) and nucleicsequence (top) of GGF2HBS5 (SEQ ID No. 167). The bottom (intermittent)sequence represents peptide sequences derived from GGF-II preparations(see FIGS. 11, 12).

FIG. 46 is a graph depicting the Schwann cell mitogenic activity ofrecombinant human and bovine glial growth factors.

FIG. 47 is a dose-response curve depicting Schwann cell proliferationactivity data resulting from administration of different size aliquotsof CHO cell conditioned medium.

FIG. 48 is a dose-response curve depicting Schwann cell mitogenicactivity secreted into the extracellular medium by SF9 insect cellsinfected with baculovirus containing the GGF2HBS5 cDNA clone.

FIG. 49 iS a Western blot of recombinant CHO cell conditioned mediumusing a GGF peptide antibody.

FIG. 50 (A) is a graph of Schwann cell proliferation activity ofrecombinant (COS cell produced) human GGF-II (rhGGF-II) peak eluted fromthe cation exchange column; (B) is an immunoblot against recombinantGGFII peak using polyclonal antibody made against specific peptide ofrhGGFII;

FIG. 51 is a graph showing the purification of rhGGF-II (CHO cellproduced) on a cation exchange column; FIG. 51(A) and 51(B) arephotographs of Western blots using the fractions of FIG. 51, and anrhGGF-II specific antibody.

FIG. 52 is a photograph of a gel depicting tyrosine phosphorylation inSchwann cells treated with recombinant glial growth factors.

FIG. 53 is the sequences of GGFHBS5, GGFHFB1 and GGFBPP5 polypeptides(SEQ ID NOS: 170, 171, and 172).

FIG. 54 is a map of the CHO cell-expression vector pcDHFRpolyA.

DETAILED DESCRIPTION

The invention pertains to the isolation and purification of novel GlialGrowth factors and the cloning of DNA sequences encoding these factors.Other components of the invention are several gene splicing variantswhich potentially encode a series of glial growth factors, in particularthe GGF2HBS5 in particular a variant which encodes the human equivalentof bovine GGF-II. It is evident that the gene encoding GGF's andp185^(erbB2) binding proteins produces a number of variably-sized,differentially-spliced RNA transcripts that give rise to a series ofproteins, which are of different lengths and contain some common peptidesequences and some unique peptide sequences. This is supported by thedifferentially-spliced sequences which are recoverable from bovineposterior pituitary RNA (as presented herein), human breast cancer(MDA-MB-231) (Holmes et al. Science 256: 1205 (1992) and chicken brainRNA (Falls et al. Cell 72: 1-20 (1993)). Further support derives fromthe wide size range of proteins which act as both mitogens for Schwanncells (as disclosed herein) and as ligands for the p185^(erbB2) receptor(see below).

Further evidence to support the fact that the genes encoding GGF andp185^(erbB2) are homologous comes from nucleotide sequence comparison.Science, 256 (1992), 1205-1210) Holmes et al. demonstrate thepurification of a 45-kilodalton human protein (Heregulin-α) whichspecifically interacts with the receptor protein p185^(erbB2), which isassociated with several human malignancies. Several complementary DNAclones encoding Heregulin-α were isolated. Peles et al. (Cell 69: 205(1992)) and Wen et al (Cell 69: 559 (1992)) describe a complementary DNAisolated from rat cells encoding a protein called "neu differentiationfactor" (NDF). The translation product of the NDF cDNA has p185^(erbB2)binding activity. Usdin and Fischbach, J. Cell. Biol. 103: 493-507(1986); Falls et al., Cold Spring Harbor Symp. Quant. Biol. 55: 397-406(1990); Harris et al., Proc. Natl. Acad. Sci. USA 88: 7664-7668 (1991);and Falls et al., Cell 72: 801-815 (1993) demonstrate the purificationof a 42 Kd glycoprotein which interacts with a receptor proteinp185^(erbB2) and several complementary cDNA clones were isolated (Fallset al. Cell 72: 801-815 (1993). Several other groups have reported thepurification of proteins of various molecular weights with p185^(erbB2)binding activity. These groups include Lupu et al. (1992) Proc. Natl.Acad. Sci. USA 89: 2287; Yarden and Peles (1991) Biochemistry 30: 3543;Lupu et al. (1990) Science 249: 1552); Dobashi et al. (1991) Biochem.Biophys. Res. Comm. 179: 1536; and Huang et al. (1992) J. Biol. Chem.257: 11508-11512.

Other Embodiments

The invention includes any protein which is substantially homologous tothe coding segments in FIG. 31 (SEQ ID Nos. 136-147, 160, and 161) aswell as other naturally occurring GGF polypeptides. Also included are:allelic variations; natural mutants; induced mutants; proteins encodedby DNA that hybridizes under high or low stringency conditions to anucleic acid naturally occurring (for definitions of high and lowstringency see Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1989, 6.3.1-6.3.6, hereby incorporated by reference);and polypeptides or proteins specifically bound by antisera to GGFpolypeptide. The term also includes chimeric polypeptides that includethe GGF polypeptides comprising sequences from FIG. 31.

The following examples are not intended to limit the invention, but areprovided to usefully illustrate the same, and provide specific guidancefor effective preparative techniques.

As will be seen from Example 3, below, the present factors exhibitmitogenic activity on a range of cell types. The activity in relation tofibroblasts indicates a wound repair ability, and the inventionencompasses this use. The general statements of invention above inrelation to formulations and/or medicaments and their manufacture shouldclearly be construed to include appropriate products and uses. This isclearly a reasonable expectation for the present invention, givenreports of similar activities for fibroblast growth factors (FGFs).Reference can be made, for example, to Sporn et al., "Peptide GrowthFactors and their Receptors I", page 396 (Baird and Bohlen) in thesection headed "FGFs in Wound Healing and Tissue Repair".

EXAMPLE 1 Purification of GGF-I and GGF-II from bovine Pituitaries

I. Preparation of Factor-CM Fraction

4,000 frozen whole bovine pituitaries (c.a. 12 kg) were thawedovernight, washed briefly with water and then homogenized in an equalvolume of 0.15M ammonium sulphate in batches in a Waring Blender. Thehomogenate was taken to pH 4.5 with 1.0M HCl and centrifuged at 4,900 gfor 80 minutes. Any fatty material in the supernatant was removed bypassing it through glass wool. After taking the pH of the supernatant to6.5 using 1.0M NaOH, solid ammonium sulphate was added to give a 36%saturated solution. After several hours stirring, the suspension wascentrifuged at 4,900 g for 80 minutes and the precipitate discarded.After filtration through glass wool, further solid ammonium sulphate wasadded to the supernatant to give a 75% saturated solution which was onceagain centrifuged at 4,900 g for 80 minutes after several hoursstirring. The pellet was resuspended in c.a. 2 L of 0.1M sodiumphosphate pH 6.0 and dialyzed against 3×40 L of the same buffer. Afterconfirming that the conductivity of the dialysate was below 20.0μSiemens, it was loaded onto a Bioprocess column (120×113 mm, Pharmacia)packed with carboxymethyl cellulose (CM-52, Whatman) at a flow rate of 2ml min⁻¹. The column was washed with 2 volumes of 0.1M sodium phosphatepH 6.0, followed by 2 volumes of 50 mM NaCl, and finally 2 volumes of0.2M NaCl both in the same buffer. During the final step, 10 mL (5minute) fractions were collected. Fractions 73 to 118 inclusive werepooled, dialyzed against 10 volumes of 10 mM sodium phosphate pH 6.0twice and clarified by centrifugation at 100,000 g for 60 minutes.

II. Hydroxylapatite HPLC

Hydroxylapatite HPLC is not a technique hitherto used in isolating glialgrowth factors, but proved particularly efficacious in this invention.The material obtained from the above CM-cellulose chromatography wasfiltered through a 0.22 μm filter (Nalgene), loaded at room temperatureon to a high performance hydroxylapatite column (50×50 mm, Biorad)equipped with a guard column (15×25 mm, Biorad) and equilibrated with 10mM potassium phosphate pH 6.0. Elution at room temperature was carriedout at a flow rate of 2 ml.minute⁻¹ using the following programmedlinear gradient:

    ______________________________________                                        time (min)                                                                            % B                                                                   ______________________________________                                        0.0      0     Solvent A: 10 mM potassium phosphate pH 6.0                    5.0      0     Solvent B: 1.0M potassium phosphate pH 6.0                     7.0     20                                                                    70.0    20                                                                    150.0   100                                                                   180.0   100                                                                   185.0    0                                                                    ______________________________________                                    

6.0 mL (3 minutes) fractions were collected during the gradient elution.Fractions 39-45 were pooled and dialyzed against 10 volumes of 50 mMsodium phosphate pH 6.0.

III. Mono S FPLC

Mono S FPLC enabled a more concentrated material to be prepared forsubsequent gel filtration.

Any particulate material in the pooled material from the hydroxylapatitecolumn was removed by a clarifying spin at 100,000 g for 60 minutesprior to loading on to a preparative HR10/10 Mono S cation exchangecolumn (100×10 mm, Pharmacia) which was then re-equilibrated to 50 mMsodium phosphate pH 6.0 at room temperature with a flow rate of 1.0ml/minute⁻¹. Under these conditions, bound protein was eluted using thefollowing programmed linear gradient:

    ______________________________________                                        time                                                                          (min) % B                                                                     ______________________________________                                        0.0    0      Solvent A:                                                                              50 mM potassium phosphate pH 6.0                      70.0   30     Solvent B:                                                                              1.2 M sodium chloride, 50 mm                          240.0 100               sodium phosphate pH 6.0                               250.0 100                                                                     260.0  0                                                                      ______________________________________                                    

1 mL (1 minute) fractions were collected throughout this gradientprogram. Fractions 99 to 115 inclusive were pooled.

IV. Gel Filtration FPLC

This step commenced the separation of the two factors of the inventionprior to final purification, producing enriched fractions.

For the purposes of this step, a preparative Superose 12 FPLC column(510×20 mm, Pharmacia) was packed according to the manufacturers'instructions. In order to standardize this column, a theoretical platesmeasurement was made according to the manufacturers' instructions,giving a value of 9,700 theoretical plates.

The pool of Mono S eluted material was applied at room temperature in2.5 Ml aliquots to this column in 50 mM sodium phosphate, 0.75 NaCl pH6.0 (previously passed through a C18 reversed phase column (Sep-pak,Millipore) at a flow rate of 1.0 mL/minute⁻¹. 1 mL (0.5 minute)fractions were collected from 35 minutes after each sample was appliedto the column. Fractions 27 to 41 (GGF-II) and 42 to 57 (GGF-I)inclusive from each run were pooled.

V. Reversed-Phase HPLC

The GGF-I and GGF-II pools from the above Superose 12 runs were eachdivided into three equal aliquots. Each aliquot was loaded on to a C8reversed-phase column (Aquapore RP-300 7 μC8 220×4.6 mm, AppliedBiosystems) protected by a guard cartridge (RP-8, 15×3.2 mm, AppliedBiosystems) and equilibrated to 40° C. at 0.5 mL.minute. Protein waseluted under these conditions using the following programmed lineargradient:

    ______________________________________                                        time (min)                                                                            % B                                                                   ______________________________________                                        0               Solvent A: 0.1% trifluoroacetic acid (TFA)                    60      66.6    Solvent B: 90% acetonitrile, 0.1% TFA                         62.0    100                                                                   72.0    100                                                                   75.0    0                                                                     ______________________________________                                    

200 μL (0.4 minute) fractions were collected in siliconized tubes(Multilube tubes, Bioquote) from 15.2 minutes after the beginning of theprogrammed gradient.

VI. SDS-Polyacrylamide Gel Electrophoresis

In this step, protein molecular weight standards, low range, catalogueno. 161-0304, from Bio-Rad Laboratories Limited, Watford, England wereemployed. The actual proteins used, and their molecular weightstandards, have been listed herein previously.

Fractions 47 to 53 (GGF-I) and fractions 61 to 67 (GGFII) inclusive fromthe reversed-phase runs were individually pooled. 7 μL of the pooledmaterial was boiled in an equal volume of 0.0125M Tris-Cl, 4% SDS, 20%glycerol, and 10% β-mercaptoethanol for GGF-I, for 5 minutes and loadedon to an 11% polyacrylamide Laemmli gel with a 4% stacking gel and runat a constant voltage of 50 V for 16 hours. This gel was then fixed andstained using a silver staining kit (Amersham). Under these conditions,the factors are each seen as a somewhat diffuse band at relativemolecular weights 30,000 to 36,000 Daltons (GGF-I) and 55,000 to 63,000Daltons (GGFII) as defined by molecular weight markers. From the gelstaining, it is apparent that there area small number of other proteinspecies present at equivalent levels to the GGF-I and GGF-II species inthe material pooled from the reversed-phase runs.

VII. Stability in Trifluoroacetic Acid

Stability data were obtained for the present Factors in the presence oftrifluoroacetic acid, as follows:

GGF-I: Material from the reversed-phase HPLC, in the presence of 0.1%TFA and acetonitrile, was assayed within 12 hours of the completion ofthe column run and then after 10 weeks incubation at 40° C. Followingincubation, the GGF-I had at least 50% of the activity of that materialassayed directly off the column.

GGF-II: Material from the reversed-phase HPLC, in the presence of 0.1%TFA and acetonitrile, and stored at -20° C., was assayed after thawingand then after 4 days incubation at 40° C. Following incubation, theGGF-II had at least 50% of the activity of that material freshly thawed.

It will be appreciated that the trifluoroacetic acid concentration usedin the above studies is that most commonly used for reversed-phasechromatography.

VIII. Activity Assay Conditions

Unless otherwise indicated, all operations were conducted at 37° C.,and, with reference to FIGS. 1 to 6, activity at each stage wasdetermined using the Brockes (Meth. Enz., supra) techniques with thefollowing modifications. Thus, in preparing Schwann cells, 5 μMforskolin was added in addition to DMEM (Dulbecco's modified Eagle'smedium), FCS and GGF. Cells used in the assay were fibroblast-freeSchwann cells at passage number less than 10, and these cells wereremoved from flasks with trypsin and plated into flat-bottomed 96-wellplates at 3.3 thousand cells per microwell.

[¹²⁵ I]IUdR was added for the final 24 hours after the test solutionaddition. The background (unstimulated) incorporation to each assay wasless than 100 cpm, and maximal incorporation was 20 to 200 fold overbackground depending on Schwann cell batch and passage number.

In the case of the GGF-I and GGF-II fractions from reversed-phase HPLCas described above, two dose response curves were also produced for eachfactor, using exactly the above method for one of the curves for eachfactor, and the above method modified in the assay procedure only bysubstituting foetal calf plasma for fetal calf serum to obtain the othercurve for each factor. The results are in FIGS. 7 and 8.

EXAMPLE 2 Amino acid Sequences of purified GGF-1 and GGF-II

Amino acid sequence analysis studies were performed using highlypurified bovine pituitary GGF-I and GGF-II. The conventional singleletter code was used to describe the sequences. Peptides were obtainedby lysyl endopeptidase and protease V8 digests, carried out on reducedand carboxymethylated samples, with the lysyl endopeptidase digest ofGGF-II carried out on material eluted from the 55-65 RD region of a 11%SDS-PAGE (MW relative to the above-quoted markers).

A total of 21 peptide sequences (see FIG. 9, SEQ ID Nos. 1-20, 169) wereobtained for GGF-I, of which 12 peptides (see FIG. 10, SEQ ID Nos. 1,22-29, 17, 19, and 32) are not present in current protein databases andtherefore represent unique sequences. A total of 12 peptide sequences(see FIG. 11, SEQ ID Nos. 33-44) were obtained for GGF-II, of which 10peptides (see FIG. 12, SEQ ID Nos. 45-53) are not present in currentprotein databases and therefore represent unique sequences (an exceptionis peptide GGF-II 06 which shows identical sequences in many proteinswhich are probably of no significance given the small number ofresidues). These novel sequences are extremely likely to correspond toportions of the true amino acid sequences of GGFs I and II.

Particular attention can be drawn to the sequences of GGF-I 07 andGGF-II 12, which are clearly highly related. The similarities indicatethat the sequences of these peptides are almost certainly those of theassigned GGF species, and are most unlikely to be derived fromcontaminant proteins.

In addition, in peptide GGF-II 02, the sequence X S S is consistent withthe presence of an N linked carbohydrate moiety on an asparagine at theposition denoted by X.

In general, in FIGS. 9 and 11, X represents an unknown residue denotinga sequencing cycle where a single position could not be called withcertainty either because there was more than one signal of equal size inthe cycle or because no signal was present. As asterisk denotes thosepeptides where the last amino acid called corresponds to the last aminoacid present in that peptide. In the remaining peptides, the signalstrength after the last amino acid called was insufficient to continuesequence calling to the end of that peptide. The right hand columnindicates the results of a computer database search using the GCGpackage FASTA and TFASTA programs to analyze the NBRF and EMBL sequencedatabases. The name of a protein in this column denotes identity of aportion of its sequence with the peptide amino acid sequence calledallowing a maximum of two mismatches. A question mark denotes threemismatches allowed. The abbreviations used are as follows:

    ______________________________________                                        HMG-1        High Mobility Group protein-1                                    HMG-2        High Mobility Group protein-2                                    LH-alpha     Luteinizing hormone alpha subunit                                LH-beta      Luteinizing hormone beta subunit                                 ______________________________________                                    

EXAMPLE 3 Mitogenic Activity of Purified GGF-I and GGF-II

The mitogenic activity of a highly purified sample containing both GGFsI and II was studied using a quantitative method, which allows a singlemicroculture to be examined for DNA synthesis, cell morphology, cellnumber and expression of cell antigens. This technique has been modifiedfrom a method previously reported by Muir et al., AnalyticalBiochemistry 185, 377-382, 1990. The main modifications are: 1) the useof uncoated microtiter plates, 2) the cell number per well, 3) the useof 5% Foetal Bovine Plasma (FBP) instead of 10% Foetal Calf Serum (FCS),and 4) the time of incubation in presence of mitogens andbromodeoxyuridine (BrdU), added simultaneously to the cultures. Inaddition the cell monolayer was not washed before fixation to avoid lossof cells, and the incubation time of monoclonal mouse anti-BrdU antibodyand peroxidase conjugated goat anti-mouse immunoglobulin (IgG) antibodywere doubled to increase the sensitivity of the assay. The assay,optimized for rat sciatic nerve Schwann cells, has also been used forseveral cell lines, after appropriate modifications to the cell cultureconditions.

I. Methods of Mitogenesis Testing

On day 1, purified Schwann cells were plated onto uncoated 96 wellplates in 5% FBP/Dulbecco's Modified Eagle Medium (DMEM) (5,000cells/well). On day 2, GGFs or other test factors were added to thecultures, as well as BrdU at a final concentration of 10 μm. After 48hours (day 4) BrdU incorporation was terminated by aspirating the mediumand cells were fixed with 200 μl/well of 70% ethanol for 20 min at roomtemperature. Next, the cells were washed with water and the DNAdenatured by incubation with 100 μl 2N HCl for 10 min at 37° C.Following aspiration, residual acid was neutralized by filling the wellswith 0.1M borate buffer, pH 9.0, and the cells were washed withphosphate buffered saline (PBS). Cells were then treated with 50 μl ofblocking buffer (PBS containing 0.1% Triton X 100 and 2% normal goatserum) for 15 min at 37° C. After aspiration, monoclonal mouse anti-BrdUantibody (Dako Corp., Santa Barbara, Calif.) (50 μl/well, 1.4 μg/mldiluted in blocking buffer) was added and incubated for two hours at 37°C. Unbound antibodies were removed by three washes in PBS containing0.1% Triton X-100 and peroxidase-conjugated goat ant-mouse IgG antibody(Dako Corp., Santa Barbara, Calif.) (50 μl/well, 2 μg/ml diluted inblocking buffer) was added and incubated for one hour at 37° C. Afterthree washes in PBS/Triton and a final rinse in PBS, wells received 100μl/well of 50 mM phosphate/citrate buffer, pH 5.0, containing 0.05% ofthe soluble chromogen o-phenylenediamine (OPD) and 0.02% H₂ O₂. Thereaction was terminated after 5-20 min at room temperature, by pipetting80 μl from each well to a clean plate containing 40 μl/well of 2Nsulfuric acid. The absorbance was recorded at 490 nm using a platereader (Dynatech Labs). The assay plates containing the cell monolayerswere washed twice with PBS and immunocytochemically stained for BrdU-DNAby adding 100 μl/well of the substrate diaminobenzidine (DAB) and 0.02%H₂ O₂ to generate an insoluble product. After 10-20 min the stainingreaction was stopped by washing with water, and BrdU-positive nucleiobserved and counted using an inverted microscope. Occasionally,negative nuclei were counterstained with 0.001% Toluidine blue andcounted as before.

II. Cell lines used for Mitogenesis Assays

Swiss 3T3 Fibroblasts:

Cells, from Flow Labs, were maintained in DMEM supplemented with 10%FCS, penicillin and streptomycin, at 37° C. in a humidified atmosphereof 10% CO₂ in air. Cells were fed or subcultured every two days. Formitogenic assay, cells were plated at a density of 5,000 cells/well incomplete medium and incubated for a week until cells were confluent andquiescent. The serum containing medium was removed and the cellmonolayer washed twice with serum free-medium. 100 μl of serum freemedium containing mitogens and 10 μM of BrdU were added to each well andincubated for 48 hours. Dose responses to GGFs and serum or PDGF (as apositive control) were performed.

BHK (Baby Hamster Kidney) 21 C13 Fibroblasts:

Cells from European Collection of Animal Cell Cultures (ECACC), weremaintained in Glasgow Modified Eagle Medium (GMEM) supplemented with 5%tryptose phosphate broth, 5% FCS, penicillin and streptomycin, at 37° C.in a humidified atmosphere of 5% CO₂ in air. Cells were fed orsubcultured every two to three days. For mitogenic assay, cells wereplated at a density of 2,000 cell/well in complete medium for 24 hours.The serum containing medium was then removed and after washing withserum free medium, replaced with 100 μl of 0.1% FCS containing GMEM orGMEM alone. GGFs and FCS or bFGF as positive controls were added,coincident with 10 μM BrdU, and incubated for 48 hours. Cell cultureswere then processed as described for Schwann cells.

C6 Rat Glioma Cell Line:

Cells, obtained at passage 39, were maintained in DMEM containing 5%FCS, 5% Horse serum (MS), penicillin and streptomycin, at 37° C. in ahumidified atmosphere of 10% CO₂ in air. Cells were fed or subculturedevery three days. For mitogenic assay, cells were plated at a density of2,000 cells/well in complete medium and incubated for 24 hours. Thenmedium was replaced with a mixture of 1: 1 DMEM and F12 mediumcontaining 0.1% FCS, after washing in serum free medium. Dose responsesto GGFs, FCS and αFGF were then performed and cells were processedthrough the ELISA as previously described for the other cell types.

PC12 (Rat Adrenal Pheochromocytoma Cells):

Cells from ECACC, were maintained in RPMI 1640 supplemented with 10% MS,5% FCS, penicillin and streptomycin, in collagen coated flasks, at 37° Cin a humidified atmosphere of 5% CO₂ in air. Cells were fed every threedays by replacing 80% of the medium. For mitogenic assay, cells wereplated at a density of 3,000 cells/well in complete medium, on collagencoated plates (50 μl/well collagen, Vitrogen Collagen Corp., diluted 1:50, 30 min at 37° C.) and incubated for 24 hours. The medium was thenplaced with fresh RPMI either alone or containing 1 mM insulin or 1%FCS. Dose responses to FCS/HS (1:2) as positive control and to GGFs wereperformed as before. After 48 hours cells were fixed and the ELISAperformed as previously described.

III. Results of Mitogenesis Assays:

All the experiments presented in this Example were performed using ahighly purified sample from a Sepharose 12 chromatography purificationstep (see Example 1, section D) containing a mixture of GGF-I and GGF-II(GGFs).

First, the results obtained with the BrdU incorporation assay werecompared with the classical mitogenic assay for Schwann cells based on[125]I-UdR incorporation into DNA of dividing cells, described by J. P.Brockes (Methods Enzymol. 147: 217, 1987).

FIG. 13 shows the comparison of data obtained with the two assays,performed in the same cell culture conditions (5,000 cells/well, in 5%FBP/DMEM, incubated in presence of GGFs for 48 hrs). As clearly shown,the results are comparable, but BrdU incorporation assay appears to beslightly more sensitive, as suggested by the shift of the curve to theleft of the graph, i.e. to lower concentrations of GGFS.

As described under the section "Methods of Mitogenesis Testing", afterthe immunoreactive BrdU-DNA has been quantitated by reading theintensity of the soluble product of the OPD peroxidase reaction, theoriginal assay plates containing cell monolayers can undergo the secondreaction resulting in the insoluble DAB product, which stains the BrdUpositive nuclei. The microcultures can then be examined under aninverted microscope, and cell morphology and the numbers ofBrdU-positive and negative nuclei can be observed.

In FIG. 14a and FIG. 14b the BrdU-DNA immunoreactivity, evaluated byreading absorbance at 490 nm, is compared to the number of BrdU-positivenuclei and to the percentage of BrdU-positive nuclei on the total numberof cells per well, counted in the same cultures. Standard deviationswere less than 10%. The two evaluation methods show a very goodcorrelation and the discrepancy between the values at the highest doseof GGFs can be explained by the different extent of DNA synthesis incells detected as BrdU-positive.

The BrdU incorporation essay can therefore provide additional usefulinformation about the biological activity of polypeptides on Schwanncells when compared to the (125) I-UdR incorporation assay. For example,the data reported in FIG. 15 show that GGFs can act on Schwann cells toinduce DNA synthesis, but at lower doses to increase the number ofnegative cells present in the microculture after 48 hours.

The assay has then been used on several cell lines of different origin.In FIG. 16 the mitogenic responses of Schwann cells and Swiss 3T3fibroblasts to GGFs are compared; despite the weak response obtained in3T3 fibroblasts, some clearly BrdU-positive nuclei were detected inthese cultures. Control cultures were run in parallel in presence ofseveral doses of FCS or human recombinant PDGF, showing that the cellscould respond to appropriate stimuli (not shown).

The ability of fibroblasts to respond to GGFs was further investigatedusing the BHK 21 C13 cell line. These fibroblasts, derived from kidney,do not exhibit contact inhibition or reach a quiescent state whenconfluent. Therefore the experimental conditions were designed to have avery low background proliferation without compromising the cellviability. GGFs have a significant mitogenic activity on BHK21 C13 cellsas shown by FIG. 17 and FIG. 18. FIG. 17 shows the BrdU incorporationinto DNA by BHK 21 C13 cells stimulated by GGFS in the presence of 0.1%FCS. The good mitogenic response to FCS indicates that cell cultureconditions were not limiting. In FIG. 18 the mitogenic effect of GGFs isexpressed as the number of BrdU-positive and BrdU-negative cells and asthe total number of cells counted per well. Data are representative oftwo experiments run in duplicates; at least three fields per well werecounted. As observed for Schwann cells in addition to a proliferativeeffect at low doses, GGFs also increase the numbers of nonrespondingcells surviving. The percentage of BrdU positive cells is proportionalto the increasing amounts of GGFs added to the cultures. The totalnumber of cells after 48 hours in presence of higher doses of GGFs is atleast doubled, conforming that GGFs induce DNA synthesis andproliferation in BHK21 C13 cells. Under the same conditions, cellsmaintained for 48 hours in the presence of 2% FCS showed an increase ofabout six fold (not shown).

C6 glioma cells have provided a useful model to study glial cellproperties. The phenotype expressed seems to be dependent on the cellpassage, the cells more closely resembling an astrocyte phenotype at anearly stage, and an oligodendrocyte phenotype at later stages (beyondpassage 70). C6 cells used in these experiments were from passage 39 topassage 52. C6 cells are a highly proliferating population, thereforethe experimental conditions were optimized to have a very low backgroundof BrdU incorporation. The presence of 0.1% serum was necessary tomaintain cell viability without significantly affecting the mitogenicresponses, as shown by the dose response to FCS (FIG. 19).

In FIG. 20 the mitogenic responses to aFGF (acidic Fibroblast growthfactor) and GGFs are expressed as the percentages of maximal BrdUincorporation obtained in the presence of FCS (8%). Values are averagesof two experiments, run in duplicates. The effect of GGFs was comparableto that of a pure preparation of aFGF. aFGF has been described as aspecific growth factor for C6 cells (Lim R. et al., Cell Regulation 1:741-746, 1990) and for that reason it was used as a positive control.The direct counting of BrdU positive and negative cells was not possiblebecause of the high cell density in the microcultures. In contrast tothe cell lines so far reported, PC12 cells did not show any evidentresponsiveness to GGFS, when treated under culture conditions in whichPC12 could respond to sera (mixture of FCS and HS as used routinely forcell maintenance). Nevertheless the number of cells plated per wellseems to affect the behavior of PC12 cells, and therefore furtherexperiments are required.

EXAMPLE 4 Isolating and Cloning a of Nucleotide Sequences encodingproteins containing GGF-I and GGF-II peptides

Isolation and cloning of the GGF-II nucleotide sequences was performedas outlined herein, using peptide sequence information and libraryscreening, and was performed as set out below. It will be appreciatedthat the peptides of FIGS. 4 and 5 can be used as the starting point forisolation and cloning of GGF-I sequences by following the techniquesdescribed herein. Indeed, FIG. 21, SEQ ID Nos. 54-88) shows possibledegenerate oligonucleotide probes for this purpose, and FIG. 23, SEQ IDNos. 90-119, lists possible PCR primers. DNA sequence and polypeptidesequence should be obtainable by this means as with GGF-II, and also DNAconstructs and expression vectors incorporating such DNA sequence, hostcells genetically altered by incorporating such constructs/vectors, andprotein obtainable by cultivating such host cells. The inventionenvisages such subject matter.

I. Design and Synthesis of oligonucleotide Probes and Primers

Degenerate DNA oligomer probes were designed by backtranslating theamino acid sequences (derived from the peptides generated from purifiedGGF protein) into nucleotide sequences. Oligomers represented either thecoding strand or the non-coding strand of the DNA sequence. When serine,arginine or leucine were included in the oligomer design, then twoseparate syntheses were prepared to avoid ambiguities. For example,serine was encoded by either TCN or AGY as in 537 and 538 or 609 and610. Similar codon splitting was done for arginine or leucine (e.g. 544,545). DNA oligomers were synthesized on a Biosearch 8750 4-column DNAsynthesizer using β-cyanoethyl chemistry operated at 0.2 micromole scalesynthesis. Oligomers were cleaved off the column (500 angstrom CpGresins) and deprotected in concentrated ammonium hydroxide for 6-24hours at 55°-60° C. Deprotected oligomers were dried under vacuum(Speedvac) and purified by electrophoresis in gels of 15% acrylamide (20mono: 1 bis), 50 mM Tris-borate-EDTA buffer containing 7M urea. Fulllength oligomers were detected in the gels by UV shadowing, then thebands were excised and DNA oligomers eluted into 1.5 mls H20 for 4-16hours with shaking. The eluate was dried, redissolved in 0.1 ml H₂ O andabsorbance measurements were taken at 260 nm.

Concentrations were determined according to the following formula:

    (A 260×units/ml) (60.6/length=×μM)

All oligomers were adjusted to 50 μM concentration by addition of H₂ O.

Degenerate probes designed as above are shown in FIG. 21, SEQ ID Nos.54-88.

PCR primers were prepared by essentially the same procedures that wereused for probes with the following modifications. Linkers of thirteennucleotides containing restriction sites were included at the 5' ends ofthe degenerate oligomers for use in cloning into vectors. DNA synthesiswas performed at I micromole scale using 1,000 angstrom CpG resins andinosine was used at positions where all four nucleotides wereincorporated normally into degenerate probes. Purifications of PCRprimers included an ethanol precipitation following the gelelectrophoresis purification.

II. Library Construction and Screening

A bovine genomic DNA library was purchased from Stratagene (CatalogueNumber: 945701). The library contained 2×10⁶ 15-20 kb Sau3Al partialbovine DNA fragments cloned into the vector lambda DashII. A bovinetotal brain CDNA library was purchased from Clonetech (Catalogue Number:BL 10139). Complementary DNA libraries were constructed (In Vitrogen;Stratagene) from mRNA prepared from bovine total brain, from bovinepituitary and from bovine posterior pituitary. In Vitrogen prepared twocDNA libraries: one library was in the vector lambda g10, the other invector pcDNAI (a plasmid library). The Stratagene libraries wereprepared in the vector lambda unizap. Collectively, the cDNA librariescontained 14 million primary recombinant phage.

The bovine genomic library was plated on i E. coli K12 host strain LE392on 23×23 cm plates (Nunc) at 150,000 to 200,000 phage plaques per plate.Each plate represented approximately one bovine genome equivalent.Following an overnight incubation at 37° C., the plates were chilled andreplicate filters were prepared according to procedures of Maniatis etal. (2: 60-81). Four plaque lifts were prepared from each plate ontouncharged nylon membranes (Pall Biodyne A or MSI Nitropure). The DNA wasimmobilized onto the membranes by cross-linking under UV light for 5minutes or, by baking at 80° C. under vacuum for two hours. DNA probeswere labelled using T4 polynucleotide kinase (New England Biolabs) withgamma 32P ATP (New England Nuclear; 6500 Ci/mmol) according to thespecifications of the suppliers. Briefly, 50 pmols of degenerate DNAoligomer were incubated in the presence of 600 μCi gamma ³² P-ATP and 5units T4 polynucleotide kinase for 30 minutes at 37° C. Reactions wereterminated, gel electrophoresis loading buffer was added and thenradiolabelled probes were purified by electrophoresis. ³² P labelledprobes were excised from gel slices and eluted into water.Alternatively, DNA probes were labelled via PCR amplification byincorporation of α-32P-dATP or α-³² P dCTP according to the protocol ofSchowalter and Sommer, Anal. Biochem 177: 90-94 (1989). Probes labelledin PCR reactions were purified by desalting on Sephadex G-150 columns.

Prehybridization and hybridization were performed in GMC buffer (0.52MNaPi, 7% SDS, 1% BSA, 1.5 mM EDTA, 0.1M NaCl 10 mg/ml tRNA). Washing wasperformed in oligowash (160 ml 1M Na₂ HPO₄, 200 ml 20% SDS, 8.0 ml 0.5MEDTA, 100 ml 5M NaCl, 3632 ml H20). Typically, 20 filters (400 sq.centimeters each) representing replicate copies of ten bovine genomeequivalents were incubated in 200 ml hybridization solution with 100pmols of degenerate oligonucleotide probe (128-512 fold degenerate).Hybridization was allowed to occur overnight at 5° C. below the minimummelting temperature calculated for the degenerate probe. The calculationof minimum melting temperature assumes 2° C. for an AT pair and 4° C.for a GC pair.

Filters were washed in repeated changes of oligowash at thehybridization temperatures four to five hours and finally, in 3.2Mtetramethylammonium chloride, 1% SDS twice for 30 min at a temperaturedependent on the DNA probe length. For 20 mers, the final washtemperature was 60° C. Filters were mounted, then exposed to X-ray film(Kodak XAR5) using intensifying screens (Dupont Cronex Lightening Plus).Usually, a three to five day film exposure at minus 80° C. wassufficient to detect duplicate signals in these library screens.Following analysis of the results, filters could be stripped andreprobed. Filters were stripped by incubating through two successivecycles of fifteen minutes in a microwave oven at full power in asolution of 1% SDS containing 10 mM EDTA pH8. Filters were taken throughat least three to four cycles of stripping and reprobing with variousprobes.

III. Recombinant Phage Isolation, Growth and DNA Preparation

These procedures followed standard protocol as described in RecombinantDNA (Maniatis et al 2: 60-2: 81).

IV. Analysis Of Isolated Clones Using DNA Digestion and Southern Blots

Recombinant Phage DNA samples (2 micrograms) were digested according toconditions recommended by the restriction endonuclease supplier (NewEngland Biolabs). Following a four hour incubation at 37° C., thereactions products were precipitated in the presence of 0.1M sodiumacetate and three volumes of ethanol. Precipitated DNA was collected bycentrifugation, rinsed in 75% ethanol and dried. All resuspended sampleswere loaded onto agarose gels (typically 1% in TAE buffer; 0.04M Trisacetate, 0.002M EDTA). Gel runs were at i volt per centimeter from 4 to20 hours. Markers included lambda Hind III DNA fragments and/or .Oslashed.X174HaeIII DNA fragments (New England Biolabs). The gels werestained with 0.5 micrograms/ml of ethidium bromide and photographed. Forsouthern blotting, DNA was first depurinated in the gel by treatmentwith 0.125N HCl, denatured in 0.5N NaOH and transferred in 20× SSC (3Msodium chloride, 0.03M sodium citrate) to uncharged nylon membranes.Blotting was done for 6 hours up to 24 hours, then the filters wereneutralized in 0.5 Tris HCl pH 7.5, 0.15M sodium chloride, then rinsedbriefly in 50 mM Tris-borate EDTA.

For cross-linking, the filters were wrapped first in transparent plasticwrap, then the DNA side exposed for five minutes to an ultravioletlight. Hybridization and washing was performed as described for libraryscreening (see section 2 of this Example). For hybridization analysis todetermine whether similar genes exist in other species slightmodifications were made. The DNA filter was purchased from Clonetech(Catalogue Number 7753-1) and contains 5 micrograms of EcoRI digestedDNA from various species per lane. The probe was labelled by PCRamplification reactions as described in section 2 above, andhybridizations were done in 80% buffer B(2 g polyvinylpyrrolidine, 2 gFicoll-400, 2 g bovine serum albumin, 50 ml 1M Tris-HCl (pH 7.5) 58 gNaCl, 1 g sodium pyrophosphate, 10 g sodium dodecyl sulfate, 950 ml H₂O) containing 10% dextran sulfate. The probes were denatured by boilingfor ten minutes then rapidly cooling in ice water. The probe was addedto the hybridization buffer at 10⁶ dpm ³² P per ml and incubatedovernight at 60° C. The filters were washed at 60° C. first in buffer Bfollowed by 2× SSC, 0.1% SDS then in 1× SSC, 0.1% SDS. For highstringency, experiments, final washes were done in 0.1× SSC, 1% SDS andthe temperature raised to 65° C.

Southern blot data were used to prepare a restriction map of the genomicclone and to indicate which subfragments hybridized to the GGF probes(candidates for subcloning).

V. Subcloning of Segments of DNA Homologous to Hybridization Probes

DNA digests (e.g. 5 micrograms) were loaded onto 1% agarose gels thenappropriate fragments excised from the gels following staining. The DNAwas purified by adsorption onto glass beads followed by elution usingthe protocol described by the supplier (Bio 101). Recovered DNAfragments (100-200 ng) were ligated into linearized dephosphorylatedvectors, e.g. pT3T7 (Ambion), which is a derivative of pUC18, using T4ligase (New England Biolabs). This vector carries the E. coli βlactamase gene, hence, transformants can be selected on platescontaining ampicillin. The vector also supplies β-galactosidasecomplementation to the host cell, therefore non-recombinants (blue) canbe detected using isopropylthiogalactoside and Bluogal (BethesdaResearch Labs). A portion of the ligation reactions was used totransform E. coli K12 XLl blue competent cells (Stratagene CatalogueNumber: 200236) and then the transformants were selected on LB platescontaining 50 micrograms per ml ampicillin. White colonies were selectedand plasmid mini preps were prepared for DNA digestion and for DNAsequence analysis. Selected clones were retested to determine if theirinsert DNA hybridized with the GGF probes.

VI. DNA Sequencing

Double stranded plasmid DNA templates were prepared from 5 ml culturesaccording to standard protocols. Sequencing was by the dideoxy chaintermination method using Sequenase 2.0 and a dideoxynucleotidesequencing kit (US Biochemical) according to the manufacturers protocol(a modification of Sanger et al. PNAS; USA 74: 5463 (1977)].Alternatively, sequencing was done in a DNA thermal cycler (PerkinElmer, model 4800) using a cycle sequencing kit (New England Biolabs;Bethesda Research Laboratories) and was performed according tomanufacturers instructions using a 5'-end labelled primer. Sequenceprimers were either those supplied with the sequencing kits or weresynthesized according to sequence determined from the clones. Sequencingreactions were loaded on and resolved on 0.4 mm thick sequencing gels of6% polyacrylamide. Gels were dried and exposed to X-Ray film. Typically,35S was incorporated when standard sequencing kits were used and a 32Pend labelled primer was used for cycle sequencing reactions. Sequenceswere read into a DNA sequence editor from the bottom of the gel to thetop (5' direction to 3') and data were analyzed using programs suppliedby Genetics Computer Group (GCG, University of Wisconsin).

VII. RNA Preparation and PCR Amplification

Open reading frames detected in the genomic DNA and which containedsequence encoding GGF peptides were extended via PCR amplification ofpituitary RNA. RNA was prepared from frozen bovine tissue (Pelfreeze)according to the guanidine neutral-CsCl procedure (Chirgwin et. al.Biochemistry 18: 5294(1979).) Polyadenylated RNA was selected byoligo-dT cellulose column chromatography (Aviv and Leder PNAS (USA) 69:1408 (1972)).

Specific DNA target sequences were amplified beginning with either totalRNA or polyadenylated RNA samples that had been converted to cDNA usingthe Perkin Elmer PCR/RNA Kit Number: N808-0017. First strand reversetranscription reactions used 1 μg template RNA and either primers ofoligo dT with restriction enzyme recognition site linkers attached orspecific antisense primers determined from cloned sequences withrestriction sites attached. To produce the second strand, the primerseither were plus strand unique sequences as used in 3' RACE reactions(Frohman et. al., PNAS (USA) 85: 8998 (1988)) or were oligo dT primerswith restriction sites attached if the second target site had been addedby terminal transferase tailing first strand reaction products with dATP(e.g. 5' race reactions, Frohman et. al., ibid). Alternatively, as inanchored PCR reactions the second strand primers were degenerate, hence,representing particular peptide sequences.

The amplification profiles followed the following general scheme: 1)five minutes soak file at 95° C.; 2) thermal cycle file of 1 minute, 95°C.; 1 minute ramped down to an annealing temperature of 45° C., 50° C.or 55° C.; maintain the annealing temperature for one minute; ramp up to72° C. over one minute; extend at 72° C. for one minute or for oneminute plus a 10 second auto extension; 3) extension cycle at 72° C.,five minutes, and; 4) soak file 4° C. for infinite time. Thermal cyclefiles (#2) usually were run for 30 cycles. A sixteen μl sample of each100 μl amplification reaction was analyzed by electrophoresis in 2%Nusieve 1% agarose gels run in TAE buffer at 4 volts per centimeter forthree hours. The gels were stained, then blotted to uncharged nylonmembranes which were probed with labelled DNA probes that were internalto the primers.

Specific sets of DNA amplification products could be identified in theblotting experiments and their positions used as a guide to purificationand reamplification. When appropriate, the remaining portions ofselected samples were loaded onto preparative gels, then followingelectrophoresis four to five slices of 0.5 mm thickness (bracketing theexpected position of the specific product) were taken from the gel. Theagarose was crushed, then soaked in 0.5 ml of electrophoresis bufferfrom 2-16 hours at 40° C. The crushed agarose was centrifuged for twominutes and the aqueous phase was transferred to fresh tubes.

Reamplification was done on five microliters (roughly 1% of the product)of the shred material using the same sets of primers and the reactionprofiles as in the original reactions. When the reamplificationreactions were completed, samples were extracted with chloroform andtransferred to fresh tubes. Concentrated restriction enzyme buffers andenzymes were added to the reactions in order to cleave at therestriction sites present in the linkers. The digested PCR products werepurified by gel electrophoresis, then subcloned into vectors asdescribed in the subcloning section above. DNA sequencing was donedescribed as above.

VIII. DNA Sequence Analysis

DNA sequences were assembled using a fragment assembly program and theamino acid sequences deduced by the GCG programs GelAssemble, Map andTranslate. The deduced protein sequences were used as a query sequenceto search protein sequence databases using WordSearch. Analysis was doneon a VAX Station 3100 workstation operating under VMS 5.1. The databasesearch was done on SwissProt release number 21 using GCG Version 7.0.

IX. Results of Cloning and Sequencing of genes encoding GGF-I and GGF-II

As indicated above, to identify the DNA sequence encoding bovine GGF-IIdegenerate oligonucleotide probes were designed from GGF-II peptidesequences. GGF-II 12 (SEQ ID No. 44), a peptide generated via lysylendopeptidase digestion of a purified GGF-II preparation (see FIGS. 11and 12) showed strong amino acid sequence homology with GGF-I 07 (SEQ IDNo. 39), a tryptic peptide generated from a purified GGF-I preparation.GGF-II 12 was thus used to create ten degenerate oligonucleotide probes(see oligos 609, 610 and 649 to 656 in FIG. 21, SEQ ID Nos. 69, 70, 71and 79, respectively). A duplicate set of filters were probed with twosets (set 1=609, 610; set 2=649-5656) of probes encoding two overlappingportions of GGF-II 12. Hybridization signals were observed, but, onlyone clone hybridized to both probe sets. The clone (designated GGF2BG1)was purified.

Southern blot analysis of DNA from the phage clone GGF2BG1 confirmedthat both sets of probes hybridized with that bovine DNA sequence, andshowed further that both probes reacted with the same set of DNAfragments within the clone. Based on those experiments a 4 kb Eco RIsub-fragment of the original clone was identified, subcloned andpartially sequenced. FIG. 22 shows the nucleotide sequence, SEQ ID No.89) and the deduced amino acid sequence of the initial DNA sequencereadings that included the hybridization sites of probes 609 and 650,and confirmed that a portion of this bovine genomic DNA encoded peptide12 (KASLADSGEYM).

Further sequence analysis demonstrated that GGF-II 12 resided on a 66amino acid open reading frame (see below) which has become the startingpoint for the isolation of overlapping sequences representing a putativebovine GGF-II gene and a cDNA.

Several PCR procedures were used to obtain additional coding sequencesfor the putative bovine GGF-II gene. Total RNA and oligo dT-selected(poly A containing) RNA samples were prepared from bovine totalpituitary, anterior pituitary, posterior pituitary, and hypothalamus.Using primers from the list shown in FIG. 23, SEQ ID Nos. 109-119,one-sided PCR reactions (RACE) were used to amplify cDNA ends in boththe 3' and 5' directions, and anchored PCR reactions were performed withdegenerate oligonucleotide primers representing additional GGF-IIpeptides. FIG. 24 summarizes the contiguous DNA structures and sequencesobtained in those experiments. From the 3' RACE reactions, threealternatively spliced cDNA sequences were produced, which have beencloned and sequenced. A 5' RACE reaction led to the discovery of anadditional exon containing coding sequence for at least 52 amino acids.Analysis of that deduced amino acid sequence revealed peptides GGF-II-6and a sequence similar to GGF-I-18 (see below). The anchored PCRreactions led to the identification of (cDNA) coding sequences ofpeptides GGF-II-1, 2, 3 and 10 contained within an additional cDNAsegment of 300 bp. The 5' limit of this segment (i.e., segment E, seeFIG. 31) is defined by the oligonucleotide which encodes peptideGGF-II-1 and which was used in the PCR reaction (additional 5' sequencedata exists as described for the human clone in Example 6). Thus thisclone contains nucleotide sequences encoding six out of the existingtotal of nine novel GGF-II peptide sequences.

The cloned gene was characterized first by constructing a physical mapof GGF2BG1 that allowed us to position the coding sequences as they werefound (see below, FIG. 25). DNA probes from the coding sequencesdescribed above have been used to identify further DNA fragmentscontaining the exons on this phage clone and to identify clones thatoverlap in both directions. The putative bovine GGF-II gene is dividedinto at least 5 coding segments. Coding segments are defined as discretelengths of DNA sequence which can be translated into polypeptidesequences using the universal genetic code. The coding segmentsdescribed in FIG. 31 and referred to in the present application are: 1)particular exons present within the GGF gene (e.g. coding segment a), or2) derived from sets of two or more exons that appear in specificsub-groups of mRNAs, where each set can be translated into the specificpolypeptide segments as in the gene products shown. The polypeptidesegments referred to in the claims are the translation products of theanalogous DNA coding segments. Only coding segments A and B have beendefined as exons and sequenced and mapped thus far. The summary of thecontiguous coding sequences identified is shown in FIG. 26. The exonsare listed (alphabetically) in the order of their discovery. It isapparent from the intron/exon boundaries that exon B may be included incDNAs that connect coding segment E and coding segment A. That is, exonB cannot be spliced out without compromising the reading frame.Therefore, we suggest that three alternative splicing patterns canproduce putative bovine GGF-II cDNA sequences 1, 2 and 3. The codingsequences of these, designated GGF2BPP1.CDS, GGF2BPP2.CDS andGGF2BPP3.CDS, respectively, are given in FIGS. 28a (SEQ ID No. 133), 28b(SEQ ID No. 134), and 28c (SEQ ID No. 135), respectively. The deducedamino acid sequence of the three cDNAs is also given in FIGS. 28a, (SEQID No. 133), 28b (SEQ ID No. 134), and 28c (SEQ ID No. 135).

The three deduced structures encode proteins of lengths 206, 281 and 257amino acids. The first 183 residues of the deduced protein sequence areidentical in all three gene products. At position 184 the clones differsignificantly. A codon for glycine GGT in GGF2BPP1 also serves as asplice donor for GGF2BPP2 and GGF2BPP3, which alternatively add on exonsC, C/D, C/D' and D or C, C/D and D, respectively, and shown in FIG. 33,SEQ ID No. 149). GGFIIBPP1 is a truncated gene product which isgenerated by reading past the coding segment A splice junction into thefollowing intervening sequence (intron). This represents coding segmentA' in FIG. 31 (SEQ ID No. 140). The transcript ends adjacent to acanonical AATAAA polyadenylation sequence, and we suggest that thistruncated gene product represents a bona fide mature transcript. Theother two longer gene products share the same 3' untranslated sequenceand polyadenylation site.

All three of these molecules contain six of the nine novel GGF-IIpeptide sequences (see FIG. 12) and another peptide is highly homologousto GGF-I-18 (see FIG. 27). This finding gives a high probability thatthis recombinant molecule encodes at least a portion of bovine GGF-II.Furthermore, the calculated isoelectric points for the three peptidesare consistent with the physical properties of GGF-I and II. Since themolecular size of GGF-II is roughly 60 kD, the longest of the threecDNAs should encode a protein with nearly one-half of the predictednumber of amino acids.

A probe encompassing the B and A exons was labelled via PCRamplification and used to screen a cDNA library made from RNA isolatedfrom bovine posterior pituitary. One clone (GGF2BPP5) showed the patternindicated in FIG. 30 and contained an additional DNA coding segment (G)between coding segments A and C. The entire nucleic acid sequence isshown in FIG. 32 (SEQ ID No. 148). The predicted. translation productfrom the longest open reading frame is 241 amino acids. A portion of asecond cDNA (GGF2BPP4) was also isolated from the bovine posteriorpituitary library using the probe described above. This clone showed thepattern indicated in FIG. 30. This clone is incomplete at the 5' end,but is a splicing variant in the sense that it lacks coding segments Gand D. BPP4 also displays a novel 3' end with regions M, K and L beyondregion C/D. The sequence of BPP4 is shown in FIG. 34 (SEQ ID No. 150).

EXAMPLE 5 GGF Sequences in Various Species

Database searching has not revealed any meaningful similarities betweenany predicted GGF translation products and known protein sequences. Thissuggests that GGF-II is the first member of a new family or superfamilyof proteins. In high stringency cross hybridization studies (DNAblotting experiments) with other mammalian DNAs we have shown, clearly,that DNA probes from this bovine recombinant molecule can readily detectspecific sequences in a variety of samples tested. A highly homologoussequence is also detected in human genomic DNA. The autoradiogram isshown in FIG. 29. The signals in the lanes containing rat and human DNArepresent the rat and human equivalents of the GGF gene, the sequencesof several cDNA's encoded by this gene have been recently reported byHolmes et al. (Science 256: 1205 (1992)) and Wen et al. (Cell 69: 559(1992)).

EXAMPLE 6 Isolation of a Human Science Encoding Human GGF2

Several human clones containing sequences from the bovine GGFII codingsegment E were isolated by screening a human cDNA library prepared frombrain stem (Stratagene catalog #935206). This strategy was pursued basedon the strong link between most of the GGF2 peptides (unique to GGF2)and the predicted peptide sequence from clones containing bovine Esegment. This library was screened as described in Example 4, Section IIusing the oligonucleotide probes 914-919 listed below. ##STR3##

Clones detected with these probes were further analyzed byhybridization. A probe derived from coding segment A (see FIG. 21),which was produced by labeling a polymerase chain reaction (PCR) productfrom segment A, was also used to screen the primary library. Severalclones that hybridized with both A and E derived probes were selectedand one particular clone, GGF2HBS5, was selected for further analysis.This clone is represented by the pattern of coding segments (EBACC/D'Das shown in FIG. 31). The E segment in this clone is the humanequivalent of the truncated bovine version of E shown in FIG. 37.GGF2HBS5 is the most likely candidate to encode GGF-II of all the"putative" GGF-II candidates described. The length of coding sequencesegment E is 786 nucleotides plus 264 bases of untranslated sequence.The predicted size of the protein encoded by GGF2HBS5 is approximately423 amino acids (approximately 45 kilodaltons, see FIG. 45, SEQ ID NO:167), which is similar to the size of the deglycosylated form of GGF-II(see Example 16). Additionally, seven of the GGF-II peptides listed inFIG. 27 have equivalent sequences which fall Within the protein sequencepredicted from region E. Peptides II-6 and II-12 are exceptions, whichfall in coding segment B and coding segment A, respectively. RNAencoding the GGF2HBS5 protein was produced in an in vitro transcriptionsystem driven by the bacteriophage T7 promoter resident in the vector(Bluescript SK [Stratagene Inc.] see FIG. 44) containing the GGF2HBS5insert. This RNA was translated in a cell free (rabbit reticulocyte)translation system and the size of the protein product was 45 Kd.Additionally, the cell-free product has been assayed in a Schwann cellmitogenic assay to confirm biological activity. Schwann cells treatedwith conditioned medium show both increased proliferation as measured byincorporation of ¹²⁵ I-Uridine and phosphorylation on tyrosine of aprotein in the 185 kilodalton range. Thus the size of the productencoded by GGF2HBS5 and the presence of DNA sequences which encode humanpeptides highly homologous to the bovine peptides shown in FIG. 12confirm that GGF2HBS5 encodes the human equivalent of bovine GGF2. Thefact that conditioned media prepared from cells transformed with thisclone elicits Schwann cell mitogenic activity confirms that theGGFIIHBS5 gene produce (unlike the BPP5 gene product) is secreted.Additionally the GGFIIBPP5 gene product seems to mediate the Schwanncell proliferation response via a receptor tyrosine kinase such asp185^(erbB2) or a closely related receptor (see Example 14).

EXAMPLE 7 Expression of Human Recombinant GGF2 in Mammalian and InsectCells

The GGF2HBS5 cDNA clone encoding human GGF2 (as described in Example 6and also referred to herein as HBS5) was cloned into vector pcDL-SRα296(Takebe et al. Mol. Cell. Biol. 8: 466-472 (1988) and COS-7 cells weretransfected in 100 mm dishes by the DEAE-dextran method (Sambrook et al.Molecular Cloning: A Laboratory Manual 2nd ed. CSH Laboratory New York(1989). Cell lysates or conditioned media from transiently expressingCOS cells were harvested at 3 or 4 days post-transfection. To preparelysates, cell monolayers were washed with PBS, scraped from the dishes,lysed by three freeze/thaw cycles in 150 μl of 0.25M Tris-HCl, pH 8.Cell debris was pelleted and the supernatant recovered. Conditionedmedia samples (7 ml.) were collected, then concentrated and bufferexchanged with 10 mM Tris, pH 7.4 using Centiprep-10 and Centricon-10units as described by the manufacturer (Amicon, Beverly, Mass.). Ratnerve Schwann cells were assayed for incorporation of DNA synthesisprecursors, as described (see Example 3). Conditioned media or celllysate samples were tested in the Schwann cell proliferation assay asdescribed in Example 3. The mitogenic activity data are shown in FIG.46. The cDNA, GGF2HBS5, encoding GGF2 directed the secretion of theprotein product to the medium. A small proportion of total activity wasdetectable inside the cells as determined by assays using cell lysates.GGF2HFB1 and GGFBPP5 cDNA's failed to direct the secretion of theproduct to the extracellular medium. GGF activity from these clones wasdetectable only in cell lysates (FIG. 46).

Recombinant GGF2 was also expressed in CHO cells. The GGF2HBS5 cDNAencoding GGF2 was cloned into the EcoRI site of vector pcdhfrpolyA (FIG.54) and transfected into the DHFR negative CHO cell line (DG44) by thecalcium phosphate coprecipitation method (Graham and Van Der Eb,Virology 52: 456-467 (1973). Clones were selected in nucleotide andnucleoside free α medium (Gibco) in 96-well plates. After 3 weeks,conditioned media samples from individual clones were screened forexpression of GGF by the Schwann cell proliferation assay as describedin Example 3. Stable clones which secreted significant levels of GGFactivity into the medium were identified. Schwann cell proliferationactivity data from different volume aliquots of CHO cell conditionedmedium were used to produce the dose response curve shown in FIG. 47(ref., Graham and Van Der Eb, Virology 52: 456, 1973). This material wasanalyzed on a Western blot probed with polyclonal antisera raisedagainst a GGF2 specific peptide. A broad band of approximately 69-90 Kd(the expected size of GGF2 extracted from pituitary and higher molecularweight glycoforms) is specifically labeled (FIG. 49, lane 12).

Recombinant GGF2 was also expressed in insect cells using Baculovirusexpression. Sf9 insect cells were infected with baculovirus containingthe GGF2HBS5 cDNA clone at a multiplicity of 3-5 (10⁶ cells/ml) andcultured in Sf900-II medium (Gibco). Schwann cell mitogenic activity wassecreted into the extracellular medium (FIG. 48). Different volumes ofinsect cell conditioned medium were tested in the Schwann cellproliferation assay in the absence of forskolin and the data used toproduce the dose response curve shown in FIG. 48.

This material was also analyzed on a Western blot (FIG. 47) probed withthe GGF II specific antibody described above. A band of 45 Kd, the sizeof deglycosylated GGF-II (see Example 16) was seen.

The methods used in this example were as follows:

Schwann cell mitogenic activity of recombinant human and bovine glialgrowth factors was determined as follows: Mitogenic responses ofcultured Schwann cells were measured in the presence of 5 μM forskolinusing crude recombinant GGF preparations obtained from transientmammalian expression experiments. Incorporation of [¹²⁵ I]-Uridine wasdetermined following an 18-24 hour exposure to materials obtained fromtransfected or mock transfected COS cells as described in the Methods.The mean and standard deviation of four sets of data are shown. Themitogenic response to partially purified native bovine pituitary GGF(carboxymethyl cellulose fraction; Goodearl et al., submitted) is shown(GGF) as a standard of one hundred percent activity.

cDNAs (FIG. 53) were cloned into pcDL-SRα296 (Takebe et al., Mol. CellBiol. 8: 466-472 (1988)), and COS-7 cells were transfected in 100 mmdishes by the DEAE-dextran method (Sambrook et al., In MolecularCloning. A Laboratory Manual, 2nd. ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989)). Cell lysates or conditionedmedia were harvested at 3 or 4 days post-transfection. To preparelysates, cell monolayers were washed with PBS, scraped from the dishes,and lysed by three freeze/thaw cycles in 150 μl of 0.25M Tris-HCl, pH 8.Cell debris was pelleted and the supernate recovered. Conditioned mediasamples (7 mls) were collected, then concentrated and buffer exchangedwith 10 mM Tris, pH 7.4 using Centriprep-10 and Centricon-10 units asdescribed by the manufacturer (Amicon, Beverly, Mass.). Rat sciaticnerve Schwann cells were assayed for incorporation of DNA synthesisprecursors, as described (Davis and Stroobant, J. Cell Biol. 110:1353-1360 (1990); Brockes et al., Brain Res. 165: 105-118 (1979)).

Western blots of recombinant CHO cell conditioned medium were performedas follows:

A recombinant CHO clone was cultured in 7 ml. of MCDB302 protein-freemedium for 3 days. 2 ml of conditioned medium was concentrated, bufferexchanged against 10 mM Tris-HCl, pH 7.4 and lyophilized to dryness. Thepellet was resuspended in SDS-PAGE sample buffer, subjected to reducingSDS gel electrophoresis and analyzed by Western blotting with a GGFpeptide antibody. A CHO control was done by using conditioned mediumfrom untransfected CHO-DG44 host and the CHO HBS5 levels were assayedusing conditioned medium from a recombinant clone.

EXAMPLE 8 Isolation of Other Human Sequences Related to Bovine GGF

The result in Examples 5 and 6 indicate that GGF related sequences fromhuman sources can also be easily isolated by using DNA probes derivedfrom bovine GGF sequences. Alternatively the procedure described byHolmes et al. (Science 256: 1205 (1992)) can be used. In this example ahuman protein (heregulin α), which binds to and activates thep185^(erbB2) receptor (and is related to GGF), is purified from a tumorcell line and the derived peptide sequence is used to produceoligonucleotide probes which were utilized to clone the cDNA's encodingheregulin. The biochemical assay for p185^(erbB2) receptor activation isdistinguished from Schwann cell proliferation. This is a similarapproach to that used in examples 1-4 for the cloning of GGF sequencesfrom pituitary cDNAs. The heregulin protein and complementary DNAs wereisolated from tumor cell lines according to the following procedures.Heregulin was purified from medium conditioned by MDA-MB-231 breastcancer cells (ATCC #HTB 26) grown on Percell Biolytica microcarrierbeads (Hyclone Labs). The medium (10 liters) was concentrated ˜25-foldby filtration through a membrane (10-kD cutoff) (Millipore) andclarified by centrifugation and filtration through a filter (0.22 μm).The filtrate was applied to a heparin Sepharose column (Pharmacia) andthe proteins were eluted with steps of 0.3, 0.6, and 0.9M NaCl inphosphate-buffered saline. Activity in the various chromatographicfractions was measured by quantifying the increase in tyrosinephosphorylation of p185^(erbB2) in MCF-7 breast tumor cells (ATCC # HTB22). MCF-7 cells were plated in 24-well Costar plates in F12 (50%)Dulbecco's minimum essential medium (50%) containing serum (10%) (10⁵cells per well), and allowed to attach for at least 24 hours. Prior toassay, cells were transferred into medium without serum for a minimum of1 hour. Column fractions (10 to 100 μl) were incubated for 30 min. at37°. Supernatants were then aspirated and the reaction was stopped bythe addition of SDS-PAGE sample buffer 100 μl). Samples were heated for5 min. at 100° C., and portions (10 to 15 μl) were applied to atris-glycine gel (4 to 20%) (Novex). After electrophoresis, proteinswere electroblotted onto a polyvinylidenedifluoride (PVDF) membrane andthen blocked with bovine serum albumin (5%) in tris-buffered salinecontaining Tween-20 (0.05%) (TBST). Blots were probed with a monoclonalantibody (1: 1000 dilution) to phosphotyrosine (Upstate Biotechnology)for a minimum of 1 hour at room temperature. Blots were washed withTBST, probed with an antibody to mouse immunoglobulin G conjugated toalkaline phosphatase (Promega) (diluted 1: 7500) for a minimum of 30min. at room temperature. Reactive bands were visualized with5-bromo-4-chloro-3-indoyl-1-phosphate and nitro-blue tetrazolium.Immunoblots were scanned with a Scan Jet Plus (Hewlett-Packard)densitometer. Signal intensities for unstimulated MCF-7 cells were 20 to30 units. Fully stimulated p185^(erbB2) yielded signals of 180 to 200units. The 0.6M NaCl pool, which contained most of the activity, wasapplied to a polyaspartic acid (PolyLC) column equilibrated in 17 mMsodium phosphate (pH 6.8) containing ethanol (30%). A linear gradientfrom 0.3M to 0.6M NaCl in the equilibration buffer was used to elutebound proteins. A peak of activity (at ˜0.45M NaCl) was furtherfractionated on a C4 reversed-phase column (SynChropak RP-4)equilibrated in buffer containing TFA (0.1%) and acetonitrile (15%).Proteins were eluted from this column with an acetonitrile gradient from25 to 40% over 60 min. Fractions (1 ml) were collected, assayed foractivity, and analyzed by SDS-PAGE on tris-glycine gels (4-20%, Novex).HPLC-purified HRG-α was digested with lysine C in SDS (0.1%), 10 mMdithiothreitol, 0.1M NH₄ HCO₃ (pH 8.0) for 20 hours at 37° C. and theresultant fragments were resolved on a Synchrom C4 column (4000A°, 0.2by 10 cm) The column was equilibrated in 0.1% TFA and eluted with a1-propanol gradient in 0.1% TFA (W. J. Henzel, J. T. Stults, C. Hsu, D.W. Aswad, J. Biol. Chem. 264, 15905 (1989)). Peaks from thechromatographic run were dried under vacuum and sequenced. One of thepeptides (eluting at ˜24% 1-propanol) gave the sequence[A]AEKEKTF[C]VNGGEXFMVKDLXNP (SEQ ID No. 162). Residues in brackets wereuncertain and an X represents a cycle in which it was not possible toidentify the amino acid. The initial yield was 8.5 pmol and the sequencedid not correspond to any known protein. Residues 1, 9, 15, and 22 werelater identified in the cDNA sequence as cysteine. Direct sequencing ofthe ˜45-kD band from a gel that had been overloaded and blotted onto aPVDF membrane revealed a low abundance sequenceXEXKE[G][R]GK[G]K[G]KKKEXGXG[K] (SEQ ID No. 163) with a very low initialyield (0.2 pmol). This corresponded to amino acid residues 2 to 22 ofheregulin-α (FIG. 31), suggesting that serine 2 is the NH₂ -terminus ofproHRG-α. Although the NH₂ terminus was blocked, it was observed thatoccasionally a small amount of a normally blocked protein may not bepost-translationally modified. The NH₂ terminal assignment was confirmedby mass spectrometry of the protein after digestion with cyanogenbromide. The COOH-terminus of the isolated protein has not beendefinitely identified; however, by mixture sequencing of proteolyticdigests, the mature sequence does not appear to extend past residue 241.Abbreviations for amino residues are: A, Ala; C, Cys; D, Asp; E, Glu; F,Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q,Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. As a source ofcDNA clones, an oligo(dT)-primed λgt10 (T. V. Huynn, R. A. Young, R. W.Davis, λgt10 and λgt11 DNA Cloning Techniques: A Practical Approach, D.Glover, Ed. (IRC Press, Oxford, (1984)) cDNA library was constructed (U.Gubler and B. J. Hoffman, Gene 25, 263 (1983)) with mRNA purified (J. M.Chirwin, A. E. Przbyla, R. J. MacDonald, W. J. Rutter, Biochemistry 18,5294 (1979)) from MDA-MB-231 cells. The following eightfold degenerateantisense deoxyoligonucleotide encoding the 13-amino acid sequenceAEKEKTFCVNGGE (SEQ ID No. 164)(13) was designed on the basis of humancodon frequency optima (R. Lathe, J. Mol. Biol. 183, 1 (1985)) andchemically synthesized: 5'-CTCGCC (G OR T) CC (A OR G) TTCAC (A OR G)CAGAAGGTCTTCTCCTTCTCAGC-3' (SEQ ID No. 165). For the purpose of probedesign a cysteine was assigned to an unknown residue in the amino acidsequence. The probe was labeled by phosphorylation and hybridized underlow-stringency conditions to the cDNA library. The proHRG-α protein wasidentified in this library. HRB-β1 cDNA was identified by probing asecond oligo(dT)-primed λgt10 library made from MDA-MB-231 cell mRNAwith sequences derived from both the 5' and 3' ends of proHRG-α. Clone13 (FIG. 2A) was a product of screening a primed(5'-CCTCGCTCCTTCTTCTTGCCCTTC-3' primer (SEQ ID No. 166); proHRG-αantisense nucleotides 33 to 56) MDA-MB-231 λgt10 library with 5' HRG-αsequence. A sequence corresponding to the 5' end of clone 13 as theprobe was used to identify proHRGβ2 and proHRGβ3 in a thirdoligo(dT)-primed λgt10 library derived from MDA-MB-231 cell mRNA. TwocDNA clones encoding each of the four HRGs were sequenced (F. Sanger, S.Milken, A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 1977]).Another cDNA designated clone 84 has an amino acid sequence identical toproHRGβ2 through amino acid 420. A stop codon at position 421 isfollowed by a different 3'-untranslated sequence.

EXAMPLE 9 Isolation of a Further Splicing Variant

The methods in Example 6 produced four closely related sequences(heregulin α, β1, β2, β3) which arise as a result of splicing variation.Peles et al. (Cell 69, 205 (1992)), and Wen et al. (Cell 69, 559 (1992))have isolated another splicing variant (from rat) using a similarpurification and cloning approach to that described in Examples 1-4 and6 involving a protein which binds to p185^(erbB2). The cDNA clone wasobtained as follows (via the purification and sequencing of ap185^(erbB2) binding protein from a transformed rat fibroblast cellline). A p185^(erbB2) binding protein was purified from conditionedmedium as follows. Pooled conditioned medium from three harvests of 500roller bottles (120 liters total) was cleared by filtration through 0.2μ filters and concentrated 31-fold with a Pelicon ultrafiltration systemusing membranes with a 20 kd molecular size cutoff. All the purificationsteps were performed by using a Pharmacia fast protein liquidchromatography system. The concentrated material was directly loaded ona column of heparin-Sepharose (150 ml, preequilibrated withphosphate-buffered saline (PBS)). The column was washed with PBScontaining 0.2M NaCl until no absorbance at 280 nm wavelength could bedetected. Bound proteins were then eluted with a continuous gradient(250 ml) of NaCl (from 0.2M to 1.0M), and 5 ml fractions were collected.Samples (0.01 ml of the collected fractions were used for thequantitative assay of the kinase stimulatory activity. Active fractionsfrom three column runs (total volume=360 ml) were pooled, concentratedto 25 ml by using a YM10 ultrafiltration membrane (Amicon, Danvers,Mass.), and ammonium sulfate was added to reach a concentration of 1.7M.After clearance by centrifugation (10,000× g, 15 min.), the pooledmaterial was loaded on a phenyl-Superose column (HR10/10, Pharmacia).The column was developed with a 45 ml gradient of (NH4)₂ SO₄ (from 1.7Mto no salt) in 0.1M Na₂ PO₄ (pH 7.4), and 2 ml fractions were collectedand assayed (0.002 ml per sample) for kinase stimulation (as describedin Example 6). The major peak of activity was pooled and dialyzedagainst 50 mM sodium phosphate buffer (pH 7.3). A Mono-S cation-exchangecolumn (HR5/5, Pharmacia) was preequilibrated with 50 mM sodiumphosphate. After loading the active material (0.884 mg of protein; 35ml), the column was washed with the starting buffer and then developedat a rate of 1 ml/min. with a gradient of NaCl. The kinase stimulatoryactivity was recovered at 0.45-0.55M salt and was spread over fourfractions of 2 ml each. These were pooled and loaded directly on a Cu⁺²chelating columns (1.6 ml, HR2/5 chelating Superose, Pharmacia). Most ofthe proteins adsorbed to the resin, but they gradually eluted with a 30ml linear gradient of ammonium chloride (0-1M). The activity eluted in asingle peak of protein at the range of 0.05 to 0.2M NH₄ Cl. Samples fromvarious steps of purification were analyzed by gel electrophoresisfollowed by silver staining using a kit from ICN (Costa Mesa, Calif.),and their protein contents were determined with a Coomassie blue dyebinding assay using a kit from Bio-Rad (Richmond, Calif.).

The p44 protein (10 μg) was reconstituted in 200 μl of 0.1M ammoniumbicarbonate buffer (pH 7.8). Digestion was conducted withL-1-tosyl-amide 2-phenylethyl chloromethyl ketone-treated trypsin(Serva) at 37° C. for 18 hr. at an enzyme-to-substrate ratio of 1:10.The resulting peptide mixture was separated by reverse-phase HPLC andmonitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d.×15 cm,300 Å) and an HP 1090 liquid chromatographic system equipped with adiode-array detector and a workstation. The column was equilibrated with0.1% trifluoroacetic acid (mobile phase A), and elution was effectedwith a linear gradient from 0%-55% mobile phase B (90% acetonitrile in0.1% trifluoroacetic acid) over 70 min. The flow rate was 0.2 ml/min.and the column temperature was controlled at 25° C. One-third aliquotsof the peptide peaks collected manually from the HPLC system werecharacterized by N-terminal sequence analysis by Edman degradation. Thefraction eluted after 27.7 min. (T27.7) contained mixed amino acidsequences and was further rechromatographed after reduction as follows:A 70% aliquot of the peptide fraction was dried in vacuo andreconstituted in 100 μl of 0.2M ammonium bicarbonate buffer (pH 7.8).DTT (final concentration 2 mM) was added to the solution, which was thenincubated at 37° C. for 30 min. The reduced peptide mixture was thenseparated by reverse-phase HPLC using a Vydac column (2.1 mm i.d.×15cm). Elution conditions and flow rat were identical to those describedabove. Amino acid sequence analysis of the peptide was performed with aModel 477 protein sequencer (Applied Biosystems, Inc., Foster City,Calif.) equipped with an on-line phenylthiohydantoin (PTH) amino acidanalyzer and a Model 900 data analysis system (Hunkapiller et al. (1986)In Methods of Protein Microcharacterization, J. E. Shively, ed.(Clifton, N.J.: Humana Press p. 223-247). The protein was loaded onto atrifluoroacetic acid-treated glass fiber disc precycled with polybreneand NaCl. The PTH-amino acid analysis was performed with a micro liquidchromatography system (Model 120) using dual syringe pumps andreverse-phase (C-18) narrow bore columns (Applied Biosystems, 2.1 mm×250mm). RNA was isolated from Rat1-EJ cells by standard procedures(Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y. (1982) and poly (A)⁺ was selected using an mRNA Separatorkit (Clontech Lab, Inc., Palo Alto, Calif.). cDNA was synthesized withthe Superscript kit (from BRL Life Technologies, Inc., Bethesda, Md.).Column-fractionated double-strand cDNA was ligated into an Sal1- andNot1-digested pJT-2 plasmid vector, a derivative of the pCD-X vector(Okayama and Berg, Mol. Cell Biol. 3: 280 (1983)) and transformed intoDH10B E. coli cells by electroporation (Dower et al., Nucl. Acids Res.16: 6127 (1988)). Approximately 5×10⁵ primary transformants werescreened with two oligonucleotide probes that were derived from theprotein sequences of the N-terminus of NDF (residues 5-24) and the T40.4tryptic peptide (residues 7-12). Their respective sequences were asfollows (N indicates all 4 nt): ##STR4## The synthetic oligonucleotideswere end-labeled with [γ-³² P]ATP with T4 polynucleotide kinase and usedto screen replicate sets of nitrocellulose filters. The hybridizationsolution contained 6× SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 2× Denhardt's solution, 50 μg/ml salmon sperm DNA, and20% formamide (for probe 1) or no formamide (for probe 2). The filterswere washed at either 50° C. with 0.5×SSC, 0.2% SDS, 2 mM EDTA (forprobe 1) or at 37° C. with 2×SSC, 0.2% SDS, 2 mM EDTA (for probe 2).Autoradiography of the filters gave ten clones that hybridized with bothprobes. These clones were purified by replating and probe hybridizationas described above. The cDNA clones were sequenced using an AppliedBiosystems 373A automated DNA sequencer and Applied Biosystems TaqDyeDeoxy™ Terminator cycle sequencing kits following the manufacture'sinstructions. In some instances, sequences were obtained using [³⁵S]dATP (Amersham) and Sequenase™ kits from U.S. Biochemicals followingthe manufacturer's instructions. Both strands of the cDNA clone 44 weresequenced by using synthetic oligonucleotides as primers. The sequenceof the most 5' 350 nt was determined in seven independent cDNA clones.The resultant clone demonstrated the pattern shown in FIG. 30 (NDF).

EXAMPLE 10 Strategies for Detecting Other Possible Splicing Variants

Alignment of the deduced amino acid sequences of the cDNA clones and PCRproducts of the bovine, and the published human (FIG. 31) and ratsequences show a high level of similarity, indicating that thesesequences are derived from homologous genes within the three species.The variable number of messenger RNA transcripts detectable at thecDNA/PCR product level is probably due to-extensive tissue-specificsplicing. The patterns obtained and shown in FIG. 30 suggests that othersplicing variants exist. A list of probable splicing variants isindicated in FIG. 37. Many of these variants can be obtained by codingsegment specific probing of cDNA libraries derived from differenttissues and by PCR experiments using primer pairs specific to particularcoding segments. Alternatively, the variants can be assembled fromspecific cDNA clones, PCR products or genomic DNA regions via cuttingand splicing techniques known to one skilled in the art. For example, arare restriction enzyme cutting site in a common coding segment (e.g.,A), can be used to connect the FBA amino terminus of GGF2BPP5 to carboxyterminal sequences of GGF2BPP1, GGFBPP2, GGFBPP3, or GGFBPP4. If thepresence or the absence of coding segment E and/or G provide benefit forcontemplated and stated uses, then these coding segments can be includedin expression constructs. These variant sequences can be expressed inrecombinant systems and the recombinant products can be assayed todetermine their level of Schwann cell mitogenic activity as well astheir ability to bind and activate the p185^(erbB2) receptor.

EXAMPLE 11 Identification of Functional Elements of GGF

The deduced structures of the family of GGF sequences indicate that thelongest forms (as represented by GGF2BPP4) encode transmembrane proteinswhere the extracellular part contains a domain which resembles epidermalgrowth factor (see Carpenter and Wahl in Peptide Growth Factors andTheir Receptors I pp. 69-133, Springer-Verlag, N.Y. 1991). The positionsof the cysteine residues in coding segments C and C/D or C/D' peptidesequence are conserved with respect to the analogous residues in theepidermal growth factor (EGF) peptide sequence (see FIG. 35, SEQ ID Nos.151-153). This suggests that the extracellular domain functions asreceptor recognition and biological activation sites. Several of thevariant forms lack the H, K, and L coding segments and thus may beexpressed as secreted, diffusible biologically active proteins. GGF DNAsequences encoding polypeptides which encompass the EGF-like domain(EGFL) can have full biological activity for stimulating glial cellmitogenic activity.

Membrane bound versions of this protein may induce Schwann cellproliferation if expressed on the surface of neurons duringembryogenesis or during nerve regeneration (where the surfaces ofneurons are intimately associated with the surfaces of proliferatingSchwann cells).

Secreted (non membrane bound) GGFs may act as classically diffusiblefactors which can interact with Schwann cells at some distance fromtheir point of secretion. Other forms may be released from intracells bysources via tissue injury and cell disruption. An example of a secretedGGF is the protein encoded by GGF2HBS5 (see example 6); this is the onlyGGF known which has been found to be directed to the exterior of thecell (example 7). Secretion is probably mediated via an N-terminalhydrophobic sequence found only in region E, which is the N-terminaldomain contained within recombinant GGF-II encoded by GGF2HBS5.

Other GGF's appear to be non-secreted (see example 6). These GGFs may beinjury response forms which are released as a consequence of tissuedamage.

Other regions of the predicted protein structure of GGF-II (encoded byGGF2HBS5) and other proteins containing regions B and A exhibitsimilarities to the human basement membrane heparin sulfate proteoglycancore protein (Kallunk, P. and Tryggvason, K., Cell Biology Vol. 116, p.559-571 (1992)). The peptide ADSGEY, which is located next to the secondcysteine of the C2 immunoglobulin fold in these GGF's, occurs in nine oftwenty-two C-2 repeats found in that basal lamina protein. This evidencestrongly suggests that these proteins may associate with matrix proteinssuch as those associated with neurons and glia, and may suggest a methodfor sequestration of glial growth factors at target sites.

EXAMPLE 12 Purification of GGFs from Recombinant Cells

In order to obtain full length or portions of GGFs to assay forbiological activity, the proteins can be overproduced using cloned DNA.Several approaches can be used. A recombinant E. coli cell containingthe sequences described above can be constructed. Expression systemssuch as pNH8a or pHH16a (Stratagene, Inc.) can be used for this purposeby following manufacturers procedures. Alternatively, these sequencescan be inserted in a mammalian expression vector and an overproducingcell line can be constructed. As an example, for this purpose DNAencoding a GGF, clone GGF2BPP5 has been expressed in both COS cells andChinese hamster ovary cells (see Example 7) (J. Biol. Chem. 263,3521-3527, (1981)). This vector containing GGF DNA sequences can betransfected into host cells using established procedures.

Transient expression can be examined or G418-resistant clones can begrown in the presence of methotrexate to select for cells that amplifythe dhfr gene (contained on the pMSXND vector) and, in the process,co-amplify the adjacent GGF protein encoding sequence. Because CHO cellscan be maintained in a totally serum-free, protein-free medium (Hamiltonand Ham, In Vitro 13, 537-547 (1977)), the desired protein can bepurified from the medium. Western analysis using the antisera producedin Example 9 can be used to detect the presence of the desired proteinin the conditioned medium of the overproducing cells.

The desired protein (rGGF-II) was purified from the medium conditionedby transiently expressing COS cells as follows. rGGF-II was harvestedfrom the conditioned medium and partially purified using Cation ExchangeChromatography (POROS-HS). The column was equilibrated with 33.3 mM MESat pH 6.0. Conditioned media was loaded at flow rate of 10 ml/min. Thepeak containing Schwann cell proliferation activity and immunoreactive(using the polyclonal antisera was against a GGFII peptide describedabove) was eluted with 50 mM Tris, 1M NaCl pH 8.0. (FIG. 50A and 50Brespectively).

rGGF-II is also expressed using a stable Chinese Hamster Ovary cellline. rGGF-II from the harvested conditioned media was partiallypurified using Cation Exchange Chromatograph (POROS-HS). The column wasequilibrated with PBS pH 7.4. Conditioned media was loaded at 10 ml/min.The peak containing the Schwann Cell Proliferative activity andimmunoreactivity (using GGFII polyclonal antisera) was eluted with 50 mMHepes, 500 mM NaCl pH 8.0. An additional peak was observed at 50 mMHepes, 1M NaCl pH 8.0 with both proliferation as well asimmunoreactivity (FIG. 51).

rGGF-II can be further purified using Hydrophobic InteractionChromatography as a high resolution step; Cation Exchange/Reverse phaseChromatography (if needed as second high resolution step); a vitalinactivation step and a DNA removal step such as Anion Exchangechromatography.

Detailed description of procedures used are as follows:

Schwann Cell Proliferation Activity of the recombinant GGF-II peakeluted from the Cation Exchange column was determined as follows:Mitogenic responses of the cultured Schwann cells were measured in thepresence of 5 μM forskolin using the peak eluted by 50 mM Tris 1M NaClpH 8.0. The peak was added at 20 1, 101 (1:10) 101 and (1: 100) 10 1.Incorporation of ¹²⁵ I-Uridine was determined and expressed as (CPM)following an 18-24 hour exposure.

An immunoblot using polyclonal antibody raised against a peptide ofGGF-II was carried out as follows: 10 μl of different fractions were runon 4-12% gradient gels. The gels were transferred on to Nitrocellulosepaper, and the nitrocellulose blots were blocked with 5% BSA and probedwith GGF-II-specific antibody (1: 250 dilution). ¹²⁵ I protein A (1:500dilution, Specific Activity=9.0/ci/g) was used as the secondaryantibody. The immunoblots were exposed to Kodax X-Ray films for 6 hours.The peak fractions eluted with 1M NaCl showed a broad immunoreactiveband at 65-90 Kd which is the expected size range for GGFII and highermolecular weight glycoforms.

GGF-II purification on cation exchange columns was performed as follows:CHO cell conditioned media expressing rGGFII was loaded on the cationexchange column at 10 ml/min. The column was equilibrated with PBS pH7.4. The elution was achieved with 50 mM Hepes 500 mM NaCl pH 8.0 and 50mM Hepes 1M NaCl pH 8.0 respectively. All fractions were analyzed usingthe Schwann cell proliferation assay (CPM) described herein. The proteinconcentration (mg/ml) was determined by the Bradford assay using BSA asthe standard.

A Western blot using 10 μl of each fraction was performed. As indicatedin FIG. 51A and 51B, immunoreactivity and the Schwann cell activityco-migrates.

The Schwann cell mitogenic assay described herein may be used to assaythe expressed product of the full length clone or any biologicallyactive portions thereof. The full length clone GGF2BPP5 has beenexpressed transiently in COS cells. Intracellular extracts oftransfected COS cells show biological activity when assayed in theSchwann cell proliferation assay described in Example 1. In addition,the full length close encoding GGF2HBS5 has been expressed stably in CHOand insect viral systems (Example 7) cells. In this case both cellextract and conditioned media show biological activity in the Schwanncell proliferation assay described in Example 1. Any member of thefamily of splicing variant complementary DNA's derived from the GGF gene(including the Heregulins) can be expressed in this manner and assayedin the Schwann cell proliferation assay by one skilled in the art.

Alternatively, recombinant material may be isolated from other variantsaccording to Wen et al. (Cell 69, 559 (1992)) who expressed the splicingvariant Neu differentiation factor (NDF) in COS-7 cells. cDNA clonesinserted in the pJT-2 eukaryotic plasmid vector are under the control ofthe SV40 early promoter, and are 3'-flanked with the SV40 terminationand polyadenylation signals. COS-7 cells were transfected with the pJT-2plasmid DNA by electroporation as follows: 6×10⁶ cells (in 0.8 ml ofDMEM and 10% FEBS) were transferred to a 0.4 cm cuvette and mixed with20 μg of plasmid DNA in 10 μl of TE solution (10 mM Tris-HCl (pH 8.0), 1mM EDTA). Electroporation was performed at room temperature at 1600 Vand 25 μF using a Bio-Rad Gene Pulser apparatus with the pulsecontroller unit set at 200 ohms. The cells were then diluted into 20 mlof DMEM, 10% FBS and transferred into a T75 flask (Falcon). After 14 hr.of incubation at 37° C., the medium was replaced with DMEM, 1% FBS, andthe incubation continued for an additional 48 hr. Conditioned mediumcontaining recombinant protein which was harvested from the cellsdemonstrated biological activity in a cell line expressing the receptorfor this protein. This cell line (cultured human breast carcinoma cellline AU 565) was treated with recombinant material. The treated cellsexhibited a morphology change which is characteristic of the activationof the erbB2 receptor. Conditioned medium of this type also can betested in the Schwann cell proliferation assay.

EXAMPLE 13 Purification and Assay of Other Proteins which bindp185^(erbB2) Receptor

I. Purification of gp30 and p70

Lupu et al. (Science 249, 1552 (1990)) and Lippman and Lupu (patentapplication number PCT/US91/03443 (1990)), hereby incorporated byreference, have purified a protein from conditioned media of a humanbreast cancer cell line MDA-MB-231, as follows.

Conditioned media collections were carried using well-known procedures.The media was concentrated 100-fold in an Amicon ultra-filtration cell(YM5 membrane) (Amicon, Danvers, Mass.). Once clarified andconcentrated, the media were stored at -20° C while consecutivecollections were made during the following days. The concentrated mediawere dialyzed using Spectra/por® 3 tubing (Spectrum Medical Industries,Los Angeles, Calif.) against 100 volumes of 0.1M acetic acid over a twoday period at 4° C. The material that precipitated during dialysis wasremoved by centrifugation at 4000 rpm for 30 min. at 4° C.; proteaseinhibitors were added. The clarified sample was then lyophilized.

Lyophilized conditioned medium was dissolved in 1M acetic acid to afinal concentration of about 25 mg/ml total protein. Insoluble materialwas removed by centrifugation at 10,000 rpm for 15 minutes. The samplewas then loaded onto a Sephadex G-100 column (XK 16, Pharmacia,Piscataway, N.J.), was equilibrated and was subjected to elution with 1Macetic acid at 4° C. with an upward flow of 30 ml/hr. 100 ng of proteinwas processed from 4 ml of 100-fold concentrated medium. Fractionscontaining 3 ml of eluate were lyophilized and resuspended in 300 μl PBSfor assay and served as a source for further purification.

Sephadex G-100 purified material was run on reversed-phase high pressureliquid chromatography (HPLC). The first step involved a steepacetonitrile gradient. Steep acetonitrile gradient and all other HPLCsteps were carried out at room temperature after equilibration of theC3-Reversed phase column with 0.05% TFA (Trifluoroacetic acid) in water(HPLC-grade). The samples were loaded and fractions were eluted with alinear gradient (0-45% acetonitrile in 0.05% TFA) at a flow rate of 1ml/min. over a 30 minute period. Absorbance was monitored at 280 nm. Oneml fractions were collected and lyophilized before analysis for EGFreceptor-competing activity.

A second HPLC step involved a shallow acetonitrile gradient. The pool ofactive fractions from the previous HPLC step was rechromatographed overthe same column. Elution was performed with a 0-18% acetonitrilegradient in 0.05% TFA over a 5 minute period followed by a linear 18-45%acetonitrile gradient in 0.05% TFA over a 30 minute period. The flowrate was 1.0 ml/min. and 1 ml fractions were collected. Human TGFα-likefactor was eluted at a 30-32% acetonitrile concentration as a singlepeak detectable by

Lupu et al. (Proc. Natl. Acad. Sci. 89, 2287 (1992)) purified anotherprotein which binds to the p185^(erbB2) receptor. This particularprotein, p75, was purified from conditioned medium used for the growthof SKBr-3 (a human breast cancer cell line) propagated in improvedEagle's medium (IMEM: GIBCO) supplemented with 10% fetal bovine serum(GIBCO). Protein p75 was purified from concentrated (100×) conditionedmedium using a p185^(erbB2) affinity column. The 94 Kilodaltonextracellular domain of p185^(erbB2) (which binds p75) was produced viarecombinant expression and was coupled to a polyacrylamidehydrazido-Sepharose affinity chromatography matrix. Following couplingthe matrix was washed extensively with ice cold 1.0M HCl and the beadswere activated with 0.5M NaNO₂. The temperature was maintained at 0° C.for 20 minutes and this was followed by filtration and washing with icecold 0.1M HCl. 500 ml of concentrated conditioned medium was run throughthe beads by gravity. The column was washed and eluted stepwise with1.0M citric acid at pH values from 4.0 to 2.0 (to allow dissociation ofthe erbB2 and p75). All fractions were desalted on Pharmacia PD10columns. Purification yielded a homogeneous polypeptide of 75 kDa at3.0-3.5 elution pH (confirmed by analysis on SDS/PAGE by silverstaining).

II. Binding of gp30 to p185^(erb) B2

The purified gp30 protein was tested in an assay to determine if itbound to p185^(erbB2). A competition assay with a monoclonal antibodyagainst p185^(erbB2). The gp30 protein displaced antibody binding top185^(erbB2) in SK-BR-3 and MDA-MB-453 cells (human breast carcinomacell lines expressing the p185^(erbB2) receptor). Schwann cellproliferation activity of gp30 can also be demonstrated by treatingSchwann cell cultures with purified gp30 using the assay proceduredescribed in Examples 1-3.

III. Binding of p75 to p185^(erbB2)

To assess whether the 75-kDa polypeptide (p75) obtained from SKBr-3conditioned medium was indeed a ligand for the erbB2 oncoprotein inSKBr-3 cells, a competition assay as described above for gp30 was used.It was found that the p75 exhibited binding activity, whereas materialfrom other chromatography fractions did not show such activity (data notshown). The flow-through material showed some binding activity. Thismight be due to the presence of shed erbB2 ECD.

IV. Other p185^(erbB2) ligands

Peles et al. (Cell 69, 205 (1992)) have also purified a 185^(erbB2)stimulating ligand from rat cells, (NDF, see Example 8 for method).Holmes et al. (Science 256, 1205 (1992)) have purified Heregulin α fromhuman cells which binds and stimulates 185^(erbB2) (see example 6).Tarakovsky et al. Oncogene 6: 218 (1991) have demonstrated bending of a25 kD polypeptide isolated from activated macrophages to the Neureceptor, a p185^(erbB2) homology, herein incorporated by reference.

VI. NDF Isolation

Yarden and Peles (Biochemistry 30, 3543 (1991)) have identified a 35kilodalton glycoprotein which will stimulate the 185^(erbB2) receptor.The protein was identified in conditioned medium according to thefollowing procedure. Rat I-EJ cells were grown to confluence in 175-cm²flasks (Falcon). Monolayers were washed with PBS and left in serum-freemedium for 10-16 h. The medium was discarded and replaced by freshserum-free medium that was collected after 3 days in culture. Theconditioned medium was cleared by low-speed centrifugation andconcentrated 100-fold in an Amicon ultrafiltration cell with a YM2membrane (molecular weight cutoff of 2000). Biochemical analyses of theneu stimulatory activity in conditioned medium indicate that the ligandis a 35-kD glycoprotein that it is heat stable but sensitive toreduction. The factor is precipitable by either high salt concentrationsor acidic alcohol. Partial purification of the molecule by selectiveprecipitation, heparin-agarose chromatography, and gel filtration indilute acid resulted in an active ligand, which is capable ofstimulating the protooncogenic receptor but is ineffective on theoncogenic neu protein, which is constitutively active. The purifiedfraction, however, retained the ability to stimulate also the relatedreceptor for EGF, suggesting that these two receptors are functionallycoupled through a bidirectional mechanism. Alternatively, the presumedligand interacts simultaneously with both receptors. The presentedbiochemical characteristic of the factor may be used to enable acompletely purified factor with which to explore these possibilities.

In other publications, Davis et al. (Biochem. Biophys. Res. Commun. 179,1536 (1991), Proc. Natl. Acad. Sci. 88, 8582 (1991) and Greene et al.,PCT patent application PCT/US91/02331 (1990)) describe the purificationof a protein from conditioned medium of a human T-cell (ATL-2) cellline.

ATL-2 cell line is an IL-2-independent HTLV-1 (+) T cell line.Mycoplasm-free ATL-2 cells were maintained in RPMI 1640 mediumcontaining 10% FCB as the culture medium (10% FCS-RPMI 1640) at 37° C.in a humidified atmosphere with 5% CO₂.

For purification of the proteinaceous substance, ATL-2 cells were washedtwice in 1×PBS and cultured at 3×10⁵ ml in serum-free RPMI 1640 medium/2mM L-glutamine for seventy-two hours followed by pelleting of the cells.The culture supernatant so produced is termed "conditioned medium"(C.M.).

C.M. was concentrated 100 fold, from 1 liter to 10 ml, using a YM-2Diaflo membrane (Amicon, Boston, Mass.) with a 1000d cutoff. For use insome assays, concentrated C.M. containing components greater than 1000MWwere rediluted to original volume with RPMI medium. Gel electrophoresisusing a polyacrylamide gradient gel (Integrated Separation Systems, HydePark, Md. or Phorecast System by Amersham, Arlington Heights, Ill.)followed by silver staining of some of this two column purified materialfrom the one liter preparation revealed at least four to five bands ofwhich the 10 kD and 20 kD bands were unique to this material. PassedC.M. containing components less than 1000 NW were used without dilution.

Concentrated conditioned medium was filter sterilized with a 0.45μuniflo filter (Schleicher and Schuell, Keene, N.H.) and then furtherpurified by application to a DEAE-SW anion exchange column (Waters,Inc., Milford, Mass.) which had been preequilibrated with 10 mM Tris-Cl,pH 8.1 Concentrated C.M. proteins representing one liter of originalATL-2 conditioned medium per HPLC run were absorbed to the column andthen eluted with a linear gradient of 0 mM to 40 mM NaCl at a flow rateof 4 ml/min. Fractions were assayed using an in vitro immune complexkinase assay with 10% of the appropriate DEAE fraction (1 columnpurified material) or 1% of the appropriate C18 fractions (two columnpurified material). The activity which increased the tyrosine kinaseactivity of p185c-neu in a dose-dependent manner using the in vitroimmune complex kinase assay was eluted as one dominant peak across 4 to5 fractions (36-40) around 220 to 240 mM of NaCl. After HPLC-DEAEpurification, the proteins in the active fractions were concentrated andpooled, concentrated and subjected to C18 (million matrix) reverse phasechromatography (Waters, Inc., Milford, Mass.) (referred to as the C18+1step or two column purified material). Elution was performed under alinear gradient of 2-propanol against 0.1% TFA. All the fractions weredialyzed against RPMI 1640 medium to remove the 2-propanol and assayedusing the in vitro immune complex kinase assay, described below, and a1% concentration of the appropriate fraction. The activity increasingthe tyrosine kinase activity of p185c-neu was eluted in two peaks. Oneeluted in fraction 11-13, while a second, slightly less active peak ofactivity eluted in fractions 20-23. These two peaks correspond to around5 to 7% of isopropanol and 11 to 14% isopropanol respectively. C18 #1generated fractions 11-13 were used in the characterization studies.Active fractions obtained from the second chromatographic step werepooled, and designated as the proteinaceous substance sample.

A twenty liter preparation employed the same purification strategy. TheDEAE active fractions 35-41 were pooled and subjected to c18chromatography as discussed above. C18#1 fractions 11-13 and 21-24 bothhad dose-dependent activity. The pool of fractions 11-13 was subjectedto an additional C18 chromatographic step (referred to as C18#2 or threecolumn purified material). Again, fractions 11-13 and 21-24 hadactivity. The dose response of fraction 23 as determined by in vitroimmune complex kinase assay as described in Example 8 may be obtainedupon addition of 0.005% by volume fraction 23 and 0.05% by volumefraction 23. This represents the greatest purity achieved.

Molecular weight ranges were determined based on gel filtrationchromatography and ultrafiltration membrane analysis. Near equal amountsof tyrosine kinase activity were retained and passed by a 10,000molecular weight cut off filter. Almost all activity was passed by a30,000 molecular weight cut off filter. Molecular weight ranges foractive chromatographic fractions were determined by comparing fractionscontaining dose-dependent neu-activating activity to the elutionprofiles of a set of protein molecular weight standards (Sigma ChemicalCo., St. Louis, Mo.) generated using the same running conditions. A lowmolecular weight region of activity was identified between 7,000 and14,000 daltons. A second range of activity ranged from about 14,000 toabout 24,000 daltons.

After gel electrophoresis using a polyacrylamide gradient gel(Integrated Separation Systems, Hyde Park, Md. or Phorecase System byAmersham, Arlington Heights, Ill.), silver staining of the three-columnpurified material (c18#2) was done with a commercially available silverstaining kit (BioRad, Rockville Centre, N.Y.). Fraction 21, 22, 23, and24 from c18#2 purification of the twenty liter preparation were run withmarkers. Fractions 22 and 23 showed the most potent dose response in the185^(erbB2) (neu) kinase assay (see below). The fact that selectedmolecular weight fractions interact with 185^(erbB2) was demonstratedwith an immune complex kinase assay.

Huang et al. (1992, J. Biol. Chem. 257: 11508-11512), herebyincorporated by reference, have isolated an additional neu/erb B2 ligandgrowth factor from bovine kidney. The 25 kD polypeptide factor wasisolated by a procedure of column fractionation, followed by sequentialcolumn chromatography on DEAE/cellulose (DE52), Sulfadex (sulfatedSephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast proteinliquid chromatography). The factor, NEL-GF, stimulates tyrosine-specificautophosphorylation of the neu/erb B2 gene product.

VII. Immune complex assay NDF for ligand binding to

p185^(erb) B2:

This assay reflects the differences in the autophosphorylation activityof immunoprecipitated p185 driven by pre-incubation of PN-NR6 celllysate with varying amounts of ATL-2 conditioned medium (C.H.) orproteinaceous substance and is referred to hereinafter as neu-activatingactivity.

Cell lines used in the immune complex kinase assay were obtained,prepared and cultured according to the methods disclosed in Kokai etal., Cell 55, 287-292 (Jul. 28, 1989) the disclosures of which arehereby incorporated by reference as if fully set forth herein, and U.S.application Ser. No. 386,820 filed Jul. 27, 1989 in the name of Mark I.Green entitled "Methods of Treating Cancerous Cells with Anti-ReceptorAntibodies", the disclosures of which are hereby incorporated byreference as if fully set forth herein.

Cell lines were all maintained in DMEM medium containing 5% FCS as theculture medium (5% FCS-DMEM) at 37° C. in a humidified atmosphere with5% CO₂.

Dense cultures of cells in 150 mm dishes were washed twice with coldPBS, scraped into 10 ml of freeze-thaw buffer (150 mM NaCl, 1 mM MgCl₂,20 mM Hepes, pH 7.2, 10% Glycerol, 1 mM EDTA, 1% Aprotinin), andcentrifuged (600×6, 10 minutes). Cell pellets were resuspended in 1 mlLysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 3% Brij 35, 1 mM EDTA,1.5 mM MgCl₂, 1% Aprotinin, 1 mM EGTA, 20 μM Na₃ VO₄, 10% Glycerol) androtated for thirty minutes at 4° C. All chemicals were from SigmaChemical Co., St. Louis, Mo., unless otherwise indicated. The insolublematerials were removed by centrifugation at 40,000× g for thirtyminutes. The clear supernatant which was subsequently used is designatedas cell lysate.

The cell lysates were incubated for fifteen minutes with 50 μl of 50%(volume/volume) Protein A-sepharose (Sigma Chemical Co., St. Louis,Mo.), and centrifugated for two minutes to preclear the lysates. 50 μlaliquots of precleared cell lysate were incubated on ice for fifteenminutes with conditioned medium, proteinaceous substance, or otherfactors as specified, in a final volume of 1 ml with lysis buffer. Thesample was then incubated with 5 μg of 7.16.4 monoclonal antibody, whichrecognizes the extracellular domain of the p185neu and p185c-neu, orother appropriate antibodies, for twenty minutes on ice, followed by atwenty minute incubation with 50 μl of 50% (vol/vol) protein A-Sepharosewith rotation at 4° C. Immune complexes were collected bycentrifugation, washed four times with 500 μl of washing buffer (50 mMHepes, pH 7.5, 0.1%, Brij 35, 150 mM NaCl, 2 mM EDTA, 1% Aprontinin, 30μm Na₃ VO₄), then twice with reaction buffer (20 mM Hepes (pH 7.4), 3 mMMnCl₂ and 0.1% Brij 35, 30 μm Na₃ VO₄). Pellets were resuspended in 50μl of reaction buffer and (Gamma-³² P]-ATP (Amersham, Arlington Heights,Ill.) was added giving a final concentration of 0.2 μm. The samples wereincubated at 27° C. for twenty minutes or at 4° C. for 25 minutes withpurer samples. The reactions were terminated by addition of 3× SDSsample buffer containing 2 mM ATP and 2 mM EDTA and then incubating themat 100° C. for five minutes. The samples were then subjected to SDS-PAGEanalysis on 10% acrylamide gels. Gels were stained, dried, and exposedto Kodak XAR or XRP film with intensifying screens.

VIII. Purification of acetylcholine receptor inducing activity (ARIA)

ARIA, a 42 kD protein which stimulates acetylcholine receptor synthesis,has been isolated in the laboratory of Gerald Fischbach (Falls et al.,Cell 72: 801-815 (1993)). ARIA induces tyrosine phosphorylation of a 185Kda muscle transmembrane protein which resembles p185^(erbB2), andstimulates acetylcholine receptor synthesis in cultured embryonicmyotubes. Sequence analysis of cDNA clones which encode ARIA shows thatARIA is a member of the GGF/erbB2 ligand group of proteins, and this ispotentially useful in the glial cell mitogenesis stimulation and otherapplications of, e.g., GGF2 described herein.

EXAMPLE 14

Protein tyrosine phosphorylation mediated by GGF in Schwann cells

Rat Schwann cells, following treatment with sufficient levels of GlialGrowth Factor to induce proliferation, show stimulation of proteintyrosine phosphorylation (FIG. 36). Varying amounts of partiallypurified GGF were applied to a primary culture of rat Schwann cellsaccording to the procedure outlined in Example 3. Schwann cells weregrown in DMEM/10% fetal calf serum/5 μM forskolin/0.5 μg per mL GGF-CM(0.5 mL per well) in poly D-lysine coated 24 well plates. Whenconfluent, the cells were fed with DMEM/10% fetal calf serum at 0.5 mLper well and left in the incubator overnight to quiesce. The followingday, the cells were fed with 0.2mL of DMEM/10% fetal calf serum and leftin the incubator for 1 hour. Test samples were then added directly tothe medium at different concentrations and for different lengths of timeas required. The cells were then lysed in boiling lysis buffer (sodiumphosphate, 5 mM, pH 6.8; SDS, 2%, β-mercapteothanol, 5%; dithiothreitol,0.1M; glycerol, 10%; Bromophenol Blue, 0.4%; sodium vanadate, 10 mM),incubated in a boiling water bath for 10 minutes and then eitheranalyzed directly or frozen at -70° C. Samples were analyzed by runningon 7.5% SDS-PAGE gels and then electroblotting onto nitrocellulose usingstandard procedures as described by Towbin et al. (1979) Proc. Natl.Acad. Sci. USA 76: 4350-4354. The blotted nitrocellulose was probed withantiphosphotyrosine antibodies using standard methods as described inKamps and Selton (1988) Oncogene 2: 305-315. The probed blots wereexposed to autoradiography film overnight and developed using a standardlaboratory processor. Densitometric measurements were carried out usingan Ultrascan XL enhanced laser densitometer (LKB). Molecular weightassignments were made relative to prestained high molecular weightstandards (Sigma). The dose responses of protein phosphorylation andSchwann cell proliferation are very similar (FIG. 36). The molecularweight of the phosphorylated band is very close to the molecular weightof p185^(erbB2). Similar results were obtained when Schwann cells weretreated with conditioned media prepared from COS cells translates withthe GGF2HBS5 clone. These results correlate well with the expectedinteraction of the GGFs with and activation of 185^(erbB2).

This experiment has been repeated with recombinant GGF-II. Conditionedmedium derived from a CHO cell line stably transformed with the GGF-IIclone (GGF2HBS5) stimulates protein tyrosine phosphorylation using theassay described above. Mock transfected CHO cells fail to stimulate thisactivity (FIG. 52).

EXAMPLE 15

Assay for Schwann cell Proliferation by Protein Factor from theMDA-MB-231 cell line.

Schwann cell proliferation is mediated by conditioned medium derivedfrom the human breast cancer cell line MDA-MB-231. On day I of theassay, 10⁴ primary rat Schwann cells were plated in 100 μl of Dulbecco'sModified Eagle's medium supplemented with 5% fetal bovine plasma perwell in a 96 well microtiter plate. On day 2 of the assay, 10 μl ofconditioned medium (from the human breast cancer cell line MDA-MB-231,cultured as described in Example 6) was added to each well of themicrotiter plate. One day 6, the number of Schwann cells per plate wasdetermined using an acid phosphatase assay (according to the procedureof Connolly et al. Anal. Biochem. 152: 136 (1986)). The plate was washedwith 100 μl of phosphate buffered saline (PBS) and 100 μl of reactionbuffer (0.1M sodium acetate, (pH 5.5)), 0.1% Triton X-100, and 10 mMp-nitrophenyl phosphate) was added per well. The plate was incubated at37° C. for two hours and the reaction was stopped by the addition of 10μl of 1N NaOH. The optical density of each sample was read in aspectrophotometer at 410 nm. A 38% stimulation of cell number overSchwann cells treated with conditioned medium from a control cell line(HS-294T, a non-producer of erbB-2 ligand) was observed. This resultshows that a protein secreted by the MI)A-MB-231 cell line (whichsecretes a p185^(erbB2) binding activity) stimulates Schwann cellproliferation.

EXAMPLE 16

N-glycosylation of GGF

The protein sequence predicted from the cDNA sequence of GGF-IIcandidate clones GGF2BPP1,2 and 3 contains a number of consensusN-glycosylation motifs. A gap in the GGFII02 peptide sequence coincideswith the asparagine residue in one of these motifs, indicating thatcarbohydrate is probably bound at this site.

N-glycosylation of the GGFs was studied by observing mobility changes onSDS-PAGE after incubation with N-glycanase, an enzyme that cleaves thecovalent linkages between carbohydrate and aspargine residues inproteins.

N-Glycanase treatment of GGF-II yielded a major band of MW 40-42 kDa anda minor band at 45-48 kDa. Activity elution experiments undernon-reducing conditions showed a single active deglycosylated species atca 45-50 kDa.

Activity elution experiments with GGF-I also demonstrate an increase inelectrophoretic mobility when treated with N-Glycanase, giving an activespecies of MW 26-28 kDa. Silver staining confirmed that there is amobility shift, although no N-deglycosylated band could be assignedbecause of background staining in the sample used.

Deposit

Nucleic acid encoding GGF-II (cDNA, GGF2HBS5) protein (Example 6) in aplasmid pBluescript 5k, under the control of the T7 promoter, wasdeposited in the American Type Culture Collection, Rockville, Md., onSep. 2, 1992, and given ATCC Accession No. 75298. Applicant acknowledgesits responsibility to replace this plasmid should it become non-viablebefore the end of the term of a patent issued hereon, and itsresponsibility to notify the ATCC of the issuance of such a patent, atwhich time the deposit will be made available to the public. Prior tothat time the deposit will be made available to the Commissioner ofPatents under the terms of 37 CFR §1.14 and 35 USC §112.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 184                                                (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      PheLysGlyAspAlaHisThrGlu                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine; Xaa in position 12 is unknown.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      XaaAlaSerLeuAlaAspGluTyrGluTyrMetXaaLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine; Xaa in position 10 is unknown.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      XaaThrGluThrSerSerSerGlyLeuXaaLeuLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      XaaLysLeuGlyGluMetTrpAlaGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      XaaLeuGlyGluLysArgAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      XaaIleLysSerGluHisAlaGlyLeuSerIleGlyAspThrAlaLys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      XaaAlaSerLeuAlaAspGluTyrGluTyrMetArgLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      XaaIleLysGlyGluHisProGlyLeuSerIleGlyAspValAlaLys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine and Xaa in position 12 is unknown.                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      XaaMetSerGluTyrAlaPhePheValGlnThrXaaArg                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     XaaSerGluHisProGlyLeuSerIleGlyAspThrAlaLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine; Xaa in position 8 is unknown.                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     XaaAlaGlyTyrPheAlaGluXaaAlaArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine; Xaa in position 7 is unknown.                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     XaaLysLeuGluPheLeuXaaAlaLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     XaaThrThrGluMetAlaSerGluGlnGlyAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     XaaAlaLysGluAlaLeuAlaAlaLeuLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     XaaPheValLeuGlnAlaLysLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     XaaLeuGlyGluMetTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     GluTyrLysCysLeuLysPheLysTrpPheLysLysAlaThrValMet                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 8 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     GluAlaLysTyrPheSerLysXaaAspAla                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 2 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     GluXaaLysPheTyrValPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     GluLeuSerPheAlaSerValArgLeuProGlyCysProProGlyVal                              151015                                                                        AspProMetValSerPheProValAlaLeu                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2003                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N in positions 31 and 32 could be                      either A or G.                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     GGAATTCCTTTTTTTTTTTTTTTTTTTCTTNNTTTTTTTTTGCCCTTATACCTCTTCGCC60                TTTCTGTGGTTCCATCCACTTCTTCCCCCTCCTCCTCCCATAAACAACTCTCCTACCCCT120               GCACCCCCAATAAATAAATAAAAGGAGGAGGGCAAGGGGGGAGGAGGAGGAGTGGTGCTG180               CGAGGGGAAGGAAAAGGGAGGCAGCGCGAGAAGAGCCGGGCAGAGTCCGAACCGACAGCC240               AGAAGCCCGCACGCACCTCGCACCATGAGATGGCGACGCGCCCCGCGCCGC291                        MetArgTrpArgArgAlaProArgArg                                                   15                                                                            TCCGGGCGTCCCGGCCCCCGGGCCCAGCGCCCCGGCTCCGCCGCCCGC339                           SerGlyArgProGlyProArgAlaGlnArgProGlySerAlaAlaArg                              10152025                                                                      TCGTCGCCGCCGCTGCCGCTGCTGCCACTACTGCTGCTGCTGGGGACC387                           SerSerProProLeuProLeuLeuProLeuLeuLeuLeuLeuGlyThr                              303540                                                                        GCGGCCCTGGCGCCGGGGGCGGCGGCCGGCAACGAGGCGGCTCCCGCG435                           AlaAlaLeuAlaProGlyAlaAlaAlaGlyAsnGluAlaAlaProAla                              455055                                                                        GGGGCCTCGGTGTGCTACTCGTCCCCGCCCAGCGTGGGATCGGTGCAG483                           GlyAlaSerValCysTyrSerSerProProSerValGlySerValGln                              606570                                                                        GAGCTAGCTCAGCGCGCCGCGGTGGTGATCGAGGGAAAGGTGCACCCG531                           GluLeuAlaGlnArgAlaAlaValValIleGluGlyLysValHisPro                              758085                                                                        CAGCGGCGGCAGCAGGGGGCACTCGACAGGAAGGCGGCGGCGGCGGCG579                           GlnArgArgGlnGlnGlyAlaLeuAspArgLysAlaAlaAlaAlaAla                              9095100105                                                                    GGCGAGGCAGGGGCGTGGGGCGGCGATCGCGAGCCGCCAGCCGCGGGC627                           GlyGluAlaGlyAlaTrpGlyGlyAspArgGluProProAlaAlaGly                              110115120                                                                     CCACGGGCGCTGGGGCCGCCCGCCGAGGAGCCGCTGCTCGCCGCCAAC675                           ProArgAlaLeuGlyProProAlaGluGluProLeuLeuAlaAlaAsn                              125130135                                                                     GGGACCGTGCCCTCTTGGCCCACCGCCCCGGTGCCCAGCGCCGGCGAG723                           GlyThrValProSerTrpProThrAlaProValProSerAlaGlyGlu                              140145150                                                                     CCCGGGGAGGAGGCGCCCTATCTGGTGAAGGTGCACCAGGTGTGGGCG771                           ProGlyGluGluAlaProTyrLeuValLysValHisGlnValTrpAla                              155160165                                                                     GTGAAAGCCGGGGGCTTGAAGAAGGACTCGCTGCTCACCGTGCGCCTG819                           ValLysAlaGlyGlyLeuLysLysAspSerLeuLeuThrValArgLeu                              170175180185                                                                  GGGACCTGGGGCCACCCCGCCTTCCCCTCCTGCGGGAGGCTCAAGGAG867                           GlyThrTrpGlyHisProAlaPheProSerCysGlyArgLeuLysGlu                              190195200                                                                     GACAGCAGGTACATCTTCTTCATGGAGCCCGACGCCAACAGCACCAGC915                           AspSerArgTyrIlePhePheMetGluProAspAlaAsnSerThrSer                              205210215                                                                     CGCGCGCCGGCCGCCTTCCGAGCCTCTTTCCCCCCTCTGGAGACGGGC963                           ArgAlaProAlaAlaPheArgAlaSerPheProProLeuGluThrGly                              220225230                                                                     CGGAACCTCAAGAAGGAGGTCAGCCGGGTGCTGTGCAAGCGGTGCGCC1011                          ArgAsnLeuLysLysGluValSerArgValLeuCysLysArgCysAla                              235240245                                                                     TTGCCTCCCCAATTGAAAGAGATGAAAAGCCAGGAATCGGCTGCAGGT1059                          LeuProProGlnLeuLysGluMetLysSerGlnGluSerAlaAlaGly                              250255260265                                                                  TCCAAACTAGTCCTTCGGTGTGAAACCAGTTCTGAATACTCCTCTCTC1107                          SerLysLeuValLeuArgCysGluThrSerSerGluTyrSerSerLeu                              270175180                                                                     AGATTCAAGTGGTTCAAGAATGGGAATGAATTGAATCGAAAAAACAAA1155                          ArgPheLysTrpPheLysAsnGlyAsnGluLeuAsnArgLysAsnLys                              185190195                                                                     CCACAAAATATCAAGATACAAAAAAAGCCAGGGAAGTCAGAACTTCGC1203                          ProGlnAsnIleLysIleGlnLysLysProGlyLysSerGluLeuArg                              200205210                                                                     ATTAACAAAGCATCACTGGCTGATTCTGGAGAGTATATGTGCAAAGTG1251                          IleAsnLysAlaSerLeuAlaAspSerGlyGluTyrMetCysLysVal                              215220225                                                                     ATCAGCAAATTAGGAAATGACAGTGCCTCTGCCAATATCACCATCGTG1299                          IleSerLysLeuGlyAsnAspSerAlaSerAlaAsnIleThrIleVal                              230235240245                                                                  GAATCAAACGCTACATCTACATCCACCACTGGGACAAGCCATCTTGTA1347                          GluSerAsnAlaThrSerThrSerThrThrGlyThrSerHisLeuVal                              250255260                                                                     AAATGTGCGGAGAAGGAGAAAACTTTCTGTGTGAATGGAGGGGAGTGC1395                          LysCysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCys                              265270275                                                                     TTCATGGTGAAAGACCTTTCAAACCCCTCGAGATACTTGTGCAAGTGC1443                          PheMetValLysAspLeuSerAsnProSerArgTyrLeuCysLysCys                              280285290                                                                     CCAAATGAGTTTACTGGTGATCGCTGCCAAAACTACGTAATGGCCAGC1491                          ProAsnGluPheThrGlyAspArgCysGlnAsnTyrValMetAlaSer                              295300305                                                                     TTCTACAGTACGTCCACTCCCTTTCTGTCTCTGCCTGAA1530                                   PheTyrSerThrSerThrProPheLeuSerLeuProGlu                                       400405410                                                                     TAGGAGCATGCTCAGTTGGTGCTGCTTTCTTGTTGCTGCATCTCCCCTCAGATTCCACCT1590              AGAGCTAGATGTGTCTTACCAGATCTAATATTGACTGCCTCTGCCTGTCGCATGAGAACA1650              TTAACAAAAGCAATTGTATTACTTCCTCTGTTCGCGACTAGTTGGCTCTGAGATACTAAT1710              AGGTGTGTGAGGCTCCGGATGTTTCTGGAATTGATATTGAATGATGTGATACAAATTGAT1770              AGTCAATATCAAGCAGTGAAATATGATAATAAAGGCATTTCAAAGTCTCACTTTTATTGA1830              TAAAATAAAAATCATTCTACTGAACAGTCCATCTTCTTTATACAATGACCACATCCTGAA1890              AAGGGTGTTGCTAAGCTGTAACCGATATGCACTTGAAATGATGGTAAGTTAATTTTGATT1950              CAGAATGTGTTATTTGTCACAAATAAACATAATAAAAGGAAAAAAAAAAAAAA2003                     (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 11 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     AlaSerLeuAlaAspGluTyrGluTyrMetXaaLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 9 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     ThrGluThrSerSerSerGlyLeuXaaLeuLys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     AlaSerLeuAlaAspGluTyrGluTyrMetArgLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 7 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     AlaGlyTyrPheAlaGluXaaAlaArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     ThrThrGluMetAlaSerGluGlnGlyAla                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     AlaLysGluAlaLeuAlaAlaLeuLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     PheValLeuGlnAlaLysLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     GluThrGlnProAspProGlyGlnIleLeuLysLysValProMetVal                              151015                                                                        IleGlyAlaTyrThr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in positions 1, 3, 17 and 19 is                    unknown.                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     XaaGluXaaLysGluGlyArgGlyLysGlyLysGlyLysLysLysGlu                              151015                                                                        XaaGlyXaaGlyLys                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     AlaGluLysGluLysThrPheCysValAsnGlyGlyGlu                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 6 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     LysLeuGluPheLeuXaaAlaLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     XaaValHisGlnValTrpAlaAlaLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine, Xaa in position 11 is unknown.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     XaaTyrIlePhePheMetGluProGluAlaXaaSerSerGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine, Xaa in position 13 is unknown.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     XaaLeuGlyAlaTrpGlyProProAlaPheProValXaaTyr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     XaaTrpPheValValIleGluGlyLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     XaaAlaSerProValSerValGlySerValGlnGluLeuValGlnArg                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine.                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     XaaValCysLeuLeuThrValAlaAlaLeuProProThr                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Lysine or                         Arginine; Xaa in position 6 is unknown.                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     XaaAspLeuLeuLeuXaaVal                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     CysThrCysGlyCysCysLysCysCysArgThrThrCysAlaCysArg                              151015                                                                        CysAlaGlyAlaAlaGlyGlyThrCysThrThrCysThrCysCysThr                              202530                                                                        ThrCysThrCysAlaGlyCys                                                         35                                                                            (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     CysCysThrCysGlyCysThrCysCysThrThrCysThrThrCysThr                              151015                                                                        ThrGlyCysCysCysThrThrCys                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     AAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCTAC60                (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     AGTACGTCCACTCCCTTTCTGTCTCTGCCTGAATAG36                                        (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 569                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     AAGGCGGAGGAGCTGTACCAGAAGAGAGTGCTGACCATAACCGGCATCTGCATCGCCCTC60                CTTGTGGTCGGCATCATGTGTGTGGTGGCCTACTGCAAAACCAAGAAACAGCGGAAAAAG120               CTGCATGACCGTCTTCGGCAGAGCCTTCGGTCTGAACGAAACAATATGATGAACATTGCC180               AATGGGCCTCACCATCCTAACCCACCCCCCGAGAATGTCCAGCTGGTGAATCAATACGTA240               TCTAAAAACGTCATCTCCAGTGAGCATATTGTTGAGAGAGAAGCAGAGACATCCTTTTCC300               ACCAGTCACTATACTTCCACAGCCCATCACTCCACTACTGTCACCCAGACTCCTAGCCAC360               AGCTGGAGCAACGGACACACTGAAAGCATCCTTTCCGAAAGCCACTCTGTAATCGTGATG420               TCATCCGTAGAAAACAGTAGGCACAGCAGCCCAACTGGGGGCCCAAGAGGACGTCTTAAT480               GGCACAGGAGGCCCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGAGAAACCCCTGAT540               TCCTACCGAGACTCTCCTCATAGTGAAAG569                                              (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     ValHisGlnValTrpAlaAlaLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 10 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     TyrIlePhePheMetGluProGluAlaXaaSerSerGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 12 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     LeuGlyAlaTrpGlyProProAlaPheProValXaaTyr                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     TrpPheValValIleGluGlyLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     AlaSerProValSerValGlySerValGlnGluLeuValGlnArg                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO: 50:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                     ValCysLeuLeuThrValAlaAlaLeuProProThr                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 51:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                     LysValHisGlnValTrpAlaAlaLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 52:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 12 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                     LysAlaSerLeuAlaAspSerGlyGluTyrMetXaaLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 53:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 5 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                     AspLeuLeuLeuXaaVal                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 54:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                     TTYAARGGNGAYGCNCAYAC20                                                        (2) INFORMATION FOR SEQ ID NO: 55:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                     CATRTAYTCRTAYTCRTCNGC21                                                       (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                     TGYTCNGANGCCATYTCNGT20                                                        (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                     TGYTCRCTNGCCATYTCNGT20                                                        (2) INFORMATION FOR SEQ ID NO: 58:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                     CCDATNACCATNGGNACYTT20                                                        (2) INFORMATION FOR SEQ ID NO: 59:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:                                     GCNGCCCANACYTGRTGNAC20                                                        (2) INFORMATION FOR SEQ ID NO: 60:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:                                     GCYTCNGGYTCCATRAARAA20                                                        (2) INFORMATION FOR SEQ ID NO: 61:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:                                     CCYTCDATNACNACRAACCA20                                                        (2) INFORMATION FOR SEQ ID NO: 62:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:                                     TCNGCRAARTANCCNGC17                                                           (2) INFORMATION FOR SEQ ID NO: 63:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:                                     GCNGCNAGNGCYTCYTTNGC20                                                        (2) INFORMATION FOR SEQ ID NO: 64:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:                                     GCNGCYAANGCYTCYTTNGC20                                                        (2) INFORMATION FOR SEQ ID NO: 65:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:                                     TTYTTNGCYTGNAGNACRAA20                                                        (2) INFORMATION FOR SEQ ID NO: 66:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:                                     TTYTTNGCYTGYAANACRAA20                                                        (2) INFORMATION FOR SEQ ID NO: 67:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:                                     TGNACNAGYTCYTGNAC17                                                           (2) INFORMATION FOR SEQ ID NO: 68:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:                                     TGNACYAAYTCYTGNAC17                                                           (2) INFORMATION FOR SEQ ID NO: 69:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:                                     CATRTAYTCNCCNGARTCNGC21                                                       (2) INFORMATION FOR SEQ ID NO: 70:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:                                     CATRTAYTCNCCRCTRTCNGC21                                                       (2) INFORMATION FOR SEQ ID NO: 71:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:                                     NGARTCNGCYAANGANGCYTT                                                         (2) INFORMATION FOR SEQ ID NO: 72:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:                                     NGARTCNGCNAGNGANGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 73:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:                                     RCTRTCNGCYAANGANGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 74:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:                                     RCTRTCNGCNAGNGANGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 75:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:                                     NGARTCNGCYAARCTNGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 76:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:                                     NGARTCNGCNAGRCTNGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 77:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 730                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:                                     GTATGTGTCAGCCATGACCACCCCGGCTCGTATGTCACCTGTAGATTTCCACACGCCAAG60                CTCCCCCAAATCGCCCCCTTCGGAAATGTCTCCACCCGTGTCCAGCATGACGGTGTCCAT120               GCCTTCCATGGCGGTCAGCCCCTTCATGGAAGAAGAGAGACCTCTACTTCTCGTGACACC180               ACCAAGGCTGCGGGAGAAGAAGTTTGACCATCACCCTCAGCAGTTCAGCTCCTTCCACCA240               CAACCCCGCGCATGACAGTAACAGCCTCCCTGCTAGCCCCTTGAGGATAGTGGAGGATGA300               GGAGTATGAAACGACCCAAGAGTACGAGCCAGCCCAAGAGCCTGTTAAGAAACTCGCCAA360               TAGCCGGCGGGCCAAAAGAACCAAGCCCAATGGCCACATTGCTAACAGATTGGAAGTGGA420               CAGCAACACAAGCTCCCAGAGCAGTAACTCAGAGAGTGAAACAGAAGATGAAAGAGTAGG480               TGAAGATACGCCTTTCCTGGGCATACAGAACCCCCTGGCAGCCAGTCTTGAGGCAACACC540               TGCCTTCCGCCTGGCTGACAGCAGGACTAACCCAGCAGGCCGCTTCTCGACACAGGAAGA600               AATCCAGGCCAGGCTGTCTAGTGTAATTGCTAACCAAGACCCTATTGCTGTATAAAACCT660               AAATAAACACATAGATTCACCTGTAAAACTTTATTTTATATAATAAAGTATTCCACCTTA720               AATTAAACAA730                                                                 (2) INFORMATION FOR SEQ ID NO: 78:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:                                     RCTRTCNGCYAARCTNGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 79:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:                                     RCTRCTNGCNAGRCTNGCYTT21                                                       (2) INFORMATION FOR SEQ ID NO: 80:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:                                     ACNACNGARATGGCTCNNGA20                                                        (2) INFORMATION FOR SEQ ID NO: 81:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:                                     ACNACNGARATGGCAGYNGA20                                                        (2) INFORMATION FOR SEQ ID NO: 82:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:                                     CAYCARGTNTGGGCNGCNAA20                                                        (2) INFORMATION FOR SEQ ID NO: 83:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:                                     TTYGTNGTNATHGARGGNAA20                                                        (2) INFORMATION FOR SEQ ID NO: 84:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:                                     AARGGNGAYGCNCAYACNGA20                                                        (2) INFORMATION FOR SEQ ID NO: 85:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:                                     GARGCNYTNGCNGCNYTNAA20                                                        (2) INFORMATION FOR SEQ ID NO: 86:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:                                     GTNGGNTCNGTNCARGARYT20                                                        (2) INFORMATION FOR SEQ ID NO: 87:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:                                     GTNGGNAGYGTNCARGARYT20                                                        (2) INFORMATION FOR SEQ ID NO: 88:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:                                     NACYTTYTTNARDATYTGNCC21                                                       (2) INFORMATION FOR SEQ ID NO: 89:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in positions 14, 23, 90, 100, 126,                 and 135 is a stop codon.                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:                                     TCTAAAACTACAGAGACTGTATTTTCATGATCATCATAGTTCTGTGAAATATA53                       AsnTyrArgAspCysIlePheMetIleIleIleValLeuXaaAsnIle                              151015                                                                        CTTAAACCGCTTTGGTCCTGATCTTGTAGGAAGTCAGAACTTCGCATT101                           LeuLysProLeuTrpSerXaaSerCysArgLysSerGluLeuArgIle                              202530                                                                        AGCAAAGCGTCACTGGCTGATTCTGGAGAATATATGTGCAAAGTGATC149                           SerLysAlaSerLeuAlaAspSerGlyGluSerMetCysLysValIle                              354045                                                                        AGCAAACTAGGAAATGACAGTGCCTCTGCCAACATCACCATTGTGGAG197                           SerLysLeuGlyAsnAspSerAlaSerAlaAsnIleArgIleValGlu                              505560                                                                        TCAAACGGTAAGAGATGCCTACTGCGTGCTATTTCTCAGTCTCTAAGA245                           SerAsnGlyLysArgCysLeuLeuArgAlaIleSerGlnSerLeuArg                              65707580                                                                      GGAGTGATCAAGGTATGTGGTCACACTTGAATCACGCAGGTGTGTGAA293                           GlyValIleLysValCysGlyHisThrXaaIleThrGlnValCysGlu                              859095                                                                        ATCTCATTGTGAACAAATAAAAATCATGAAAGGAAAACTCTATGTTTG341                           IleSerCysXaaThrAsnLysAsnHisGluArgLysThrLeuCysLeu                              100105110                                                                     AAATATCTTATGGGTCCTCCTGTAAAGCTCTTCACTCCATAAGGTGAA389                           LysTyrLeuMetGlyProProValLysLeuPheThrProXaaGlyGlu                              115120125                                                                     ATAGACCTGAAATATATATAGATTATTT417                                               IleAspLeuLysTyrIleXaaIleIle                                                   130135                                                                        (2) INFORMATION FOR SEQ ID NO: 90:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 19, 25, and 31 is                       Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:                                     CCGAATTCTGCAGGARACNCARCCNGAYCCNGG33                                           (2) INFORMATION FOR SEQ ID NO: 91:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 14, 20, 23, 29, and                     35 is Inosine.                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:                                     AAGGATCCTGCAGNGTRTANGCNCCDATNACCATNGG37                                       (2) INFORMATION FOR SEQ ID NO: 92:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 16, 21, and 24                          is Inosine. Y can be cytidine or thymidine.                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:                                     CCGAATTCTGCAGGCNGAYTCNGGNGARTAYATG34                                          (2) INFORMATION FOR SEQ ID NO: 93:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 16 and 25 is Inosine.                   Y can be cytidine or thymidine.                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:                                     CCGAATTCTGCAGGCNGAYAGYGGNGARTAYAT33                                           (2) INFORMATION FOR SEQ ID NO: 94:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 14, 15, 16, 26, and                     29 is Inosine. Y can be cytidine or thymidine.                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:                                     AAGGATCCTGCAGNNNCATRTAYTCNCCNGARTC34                                          (2) INFORMATION FOR SEQ ID NO: 95:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 14, 15, 16, and 26                      is Inosine. Y can be cytidine or thymidine.                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:                                     AAGGATCCTGCAGNNNCATRTAYTCNCCRCTRTC34                                          (2) INFORMATION FOR SEQ ID NO: 96:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 21, 28, and 31 is                       Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:                                     CCGAATTCTGCAGCAYCARGTNTGGGCNGCNAA33                                           (2) INFORMATION FOR SEQ ID NO: 97:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 31 is Inosine. Y can                     be cytidine or thymidine.                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:                                     CCGAATTCTGCAGATHTTYTTYATGGARCCNGARG35                                         (2) INFORMATION FOR SEQ ID NO: 98:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 18, 21, 24, 27, and                     33 is Inosine. Y can be cytidine or thymidine.                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:                                     CCGAATTCTGCAGGGGGNCCNCCNGCNTTYCCNGT35                                         (2) INFORMATION FOR SEQ ID NO: 99:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 21 and 24 is Inosine.                   Y can be cytidine or thymidine.                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:                                     CCGAATTCTGCAGTGGTTYGTNGTNATHGARGG33                                           (2) INFORMATION FOR SEQ ID NO: 100:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 17, 20, and 26 is                       Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:                                    AAGGATCCTGCAGYTTNGCUNGCCCANACYTGRTG35                                         (2) INFORMATION FOR SEQ ID NO: 101:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 19 is Inosine. Y can                     be cytidine or thymidine.                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:                                    AAGGATCCTGCAGGCYTCNGGYTCCATRAARAA33                                           (2) INFORMATION FOR SEQ ID NO: 102:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 16, 22, 25, 28, and                     31 is Inosine.                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:                                    AAGGATCCTGCAGACNGGRAANGCNGGNGGNCC33                                           (2) INFORMATION FOR SEQ ID NO: 103:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 17, 26, and 29 is                       Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:                                    AAGGATCCTGCAGYTTNCCYTCDATNACNACRAAC35                                         (2) INFORMATION FOR SEQ ID NO: 104:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 18 is Inosine. Y can                     be cytidine or thymidine.                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:                                    CATRTAYTCRTAYTCTCNGCAAGGATCCTGCAG33                                           (2) INFORMATION FOR SEQ ID NO: 105:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 19, 25, and 31 is                        Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:                                    CCGAATTCTGCAGAARGGNGAYGCNCAYACNGA33                                           (2) INFORMATION FOR SEQ ID NO: 106:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 3 and 18 is Inosine.                     Y can be cytidine or thymidine.                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:                                    GCNGCYAANGCYTCYTTNGCAAGGATCCTGCAG33                                           (2) INFORMATION FOR SEQ ID NO: 107:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 3, 6, 9, and 18 is                       Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:                                    GCNGCNAGNGCYTCYTTNGCAAGGATCCTGCAG33                                           (2) INFORMATION FOR SEQ ID NO: 108:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at position 3, 12, and 15 is                         Inosine. Y can be cytidine or thymidine.                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:                                    TCNGCRAARTANCCNGCAAGGATCCTGCAG30                                              (2) INFORMATION FOR SEQ ID NO: 109:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:                                    CATCGATCTGCAGGCTGATTCTGGAGAATATATGTGCA38                                      (2) INFORMATION FOR SEQ ID NO: 110:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:                                    AAGGATCCTGCAGCCACATCTCGAGTCGACATCGATT37                                       (2) INFORMATION FOR SEQ ID NO: 111:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:                                    CCGAATTCTGCAGTGATCAGCAAACTAGGAAATGACA37                                       (2) INFORMATION FOR SEQ ID NO: 112:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:                                    CATCGATCTGCAGCCTAGTTTGCTGATCACTTTGCAC37                                       (2) INFORMATION FOR SEQ ID NO: 113:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:                                    AAGGATCCTGCAGTATATTCTCCAGAATCAGCCAGTG37                                       (2) INFORMATION FOR SEQ ID NO: 114:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:                                    AAGGATCCTGCAGGCACGCAGTAGGCATCTCTTA34                                          (2) INFORMATION FOR SEQ ID NO: 115:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:                                    CCGAATTCTGCAGCAGAACTTCGCATTAGCAAAGC35                                         (2) INFORMATION FOR SEQ ID NO: 116:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:                                    CATCCCGGGATGAAGAGTCAGGAGTCTGTGGCA33                                           (2) INFORMATION FOR SEQ ID NO: 117:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:                                    ATACCCGGGCTGCAGACAATGAGATTTCACACACCTGCG39                                     (2) INFORMATION FOR SEQ ID NO: 118:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:                                    AAGGATCCTGCAGTTTGGAACCTGCCACAGACTCCT36                                        (2) INFORMATION FOR SEQ ID NO: 119:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:                                    ATACCCGGGCTGCAGATGAGATTTCACACACCTGCGTGA39                                     (2) INFORMATION FOR SEQ ID NO: 120:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:                                    HisGlnValTrpAlaAlaLysAlaAlaGlyLeuLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 121:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:                                    GlyGlyLeuLysLysAspSerLeuLeuThrValArgLeuGlyAlaAsn                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 122:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 12 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:                                    LeuGlyAlaTrpGlyProProAlaPheProValXaaTyr                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 123:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123:                                    LeuLeuThrValArgLeuGlyAlaTrpGlyHisProAlaPheProSer                              151015                                                                        CysGlyArgLeuLysGluAsp                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO: 124:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 10 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:                                    TyrIlePhePheMetGluProGluAlaXaaSerSerGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 125:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:                                    LysGluAspSerArgTyrIlePhePheMetGluProGluAlaAsnSer                              151015                                                                        SerGlyGlyProGlyArgLeu                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO: 126:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:                                    ValAlaGlySerLysLeuValLeuArgCysGluThrSerSer                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO: 127:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:                                    GluTyrLysCysLeuLysPheLysTrpPheLysLysAlaThrValMet                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 128:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128:                                    CysGluThrSerSerGluTyrSerSerLeuLysPheLysTrpPheLys                              151015                                                                        AsnGlySerGluLeuSerArgLysAsnLys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO: 129:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 12 is unknown.                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129:                                    LysAlaSerLeuAlaAspSerGlyGluTyrMetXaaLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO: 130:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:                                    GluLeuArgIleSerLysAlaSerLeuAlaAspSerGlyGluTyrMet                              151015                                                                        CysLysValIleSerLysLeu                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO: 131:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:                                    AlaSerLeuAlaAspGluTyrGluTyrMetArgLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 132:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132:                                    LeuArgIleSerLysAlaSerLeuAlaAspSerGlyGluTyrMetCys                              151015                                                                        LysValIleSerLysLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO: 133:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 744                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:                                    CCTGCAGCATCAAGTGTGGGCGGCGAAAGCCGGGGGCTTGAAGAAGGAC                             HisGlnValTrpAlaAlaLysAlaGlyGlyLeuLysLysAsp                                    1510                                                                          TCGCTG55                                                                      SerLeu                                                                        15                                                                            CTCACCGTGCGCCTGGGCGCCTGGGGCCACCCCGCCTTCCCCTCCTGC103                           LeuThrValArgLeuGlyAlaTrpGlyHisProAlaPheProSerCys                              202530                                                                        GGGCGCCTCAAGGAGGACAGCAGGTACATCTTCTTCATGGAGCCCGAG151                           GlyArgLeuLysGluAspSerArgTyrIlePhePheMetGluProGlu                              354045                                                                        GCCAACAGCAGCGGCGGGCCCGGCCGCCTTCCGAGCCTCCTTCCCCCC199                           AlaAsnSerSerGlyGlyProGlyArgLeuProSerLeuLeuProPro                              505560                                                                        TCTCGAGACGGGCCGGAACCTCAAGAAGGAGGTCAGCCGGGTGCTGTG247                           SerArgAspGlyProGluProGlnGluGlyGlyGlnProGlyAlaVal                              65707580                                                                      CAACGGTGCGCCTTGCCTCCCCGCTTGAAAGAGATGAAGAGTCAGGAG295                           GlnArgCysAlaLeuProProArgLeuLysGluMetLysSerGlnGlu                              859095                                                                        TCTGTGGCAGGTTCCAAACTAGTGCTTCGGTGCGAGACCAGTTCTGAA343                           SerValAlaGlySerLysLeuValLeuArgCysGluThrSerSerGlu                              100105110                                                                     TACTCCTCTCTCAAGTTCAAGTGGTTCAAGAATGGGAGTGAATTAAGC391                           TyrSerSerLeuLysPheLysTrpPheLysAsnGlySerGluLeuSer                              115120125                                                                     CGAAAGAACAAACCAGAAAACATCAAGATACAGAAAAGGCCGGGGAAG439                           ArgLysAsnLysProGluAsnIleLysIleGlnLysArgProGlyLys                              130135140                                                                     TCAGAACTTCGCATTAGCAAAGCGTCACTGGCTGATTCTGGAGAATAT487                           SerGluLeuArgIleSerLysAlaSerLeuAlaAspSerGlyGluTyr                              145150155160                                                                  ATGTGCAAAGTGATCAGCAAACTAGGAAATGACAGTGCCTCTGCCAAC535                           MetCysLysValIleSerLysLeuGlyAsnAspSerAlaSerAlaAsn                              165170175                                                                     ATCACCATTGTGGAGTCAAACGGTAAGAGATGCCTACTGCGTGCTATT583                           IleThrIleValGluSerAsnGlyLysArgCysLeuLeuArgAlaIle                              180185190                                                                     TCTCAGTCTCTAAGAGGAGTGATCAAGGTATGTGGTCACACT625                                 SerGlnSerLeuArgGlyValIleLysValCysGlyHisThr                                    195200205                                                                     TGAATCACGCAGGTGTGTGAAATCTCATTGTGAACAAATAAAAATCATGAAAGGAAAAAA685               AAAAAAAAAAAATCGATGTCGACTCGAGATGTGGCTGCAGGTCGACTCTAGAGGATCCC744                (2) INFORMATION FOR SEQ ID NO: 134:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1193                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:                                    CCTGCAGCATCAAGTGTGGGCGGCGAAAGCCGGGGGCTTGAAGAAGGAC                             HisGlnValTrpAlaAlaLysAlaGlyGlyLeuLysLysAsp                                    1510                                                                          TCGCTG55                                                                      SerLeu                                                                        15                                                                            CTCACCGTGCGCCTGGGCGCCTGGGGCCACCCCGCCTTCCCCTCCTGC103                           LeuThrValArgLeuGlyAlaTrpGlyHisProAlaPheProSerCys                              202530                                                                        GGGCGCCTCAAGGAGGACAGCAGGTACATCTTCTTCATGGAGCCCGAG151                           GlyArgLeuLysGluAspSerArgTyrIlePhePheMetGluProGlu                              354045                                                                        GCCAACAGCAGCGGCGGGCCCGGCCGCCTTCCGAGCCTCCTTCCCCCC199                           AlaLysSerSerGlyGlyProGlyArgLeuProSerLeuLeuProPro                              505560                                                                        TCTCGAGACGGGCCGGAACCTCAAGAAGGAGGTCAGCCGGGTGCTGTG247                           SerArgAspGlyProGluProGlnGluGlyGlyGlnProGlyAlaVal                              65707580                                                                      CAACGGTGCGCCTTGCCTCCCCGCTTGAAAGAGATGAAGAGTCAGGAG295                           GlnArgCysAlaLeuProProArgLeuLysGluMetLysSerGlnGlu                              859095                                                                        TCTGTGGCAGGTTCCAAACTAGTGCTTCGGTGCGAGACCAGTTCTGAA343                           SerValAlaGlySerLysLeuValLeuArgCysGluThrSerSerGlu                              100105110                                                                     TACTCCTCTCTCAAGTTCAAGTGGTTCAAGAATGGGAGTGAATTAAGC391                           TyrSerSerLeuLysPheLysTrpPheLysAsnGlySerGluLeuSer                              115120125                                                                     CGAAAGAACAAACCAGAAAACATCAAGATACAGAAAAGGCCGGGGAAG439                           ArgLysAsnLysGlyGlyAsnIleLysIleGlnLysArgProGlyLys                              130135140                                                                     TCAGAACTTCGCATTAGCAAAGCGTCACTGGCTGATTCTGGAGAATAT487                           SerGluLeuArgIleSerLysAlaSerLeuAlaAspSerGlyGluTyr                              145150155160                                                                  ATGTGCAAAGTGATCAGCAAACTAGGAAATGACAGTGCCTCTGCCAAC535                           MetCysLysValIleSerLysLeuGlyAsnAspSerAlaSerAlaAsn                              165170175                                                                     ATCACCATTGTGGAGTCAAACGCCACATCCACATCTACAGCTGGGACA583                           IleThrIleValGluSerAsnAlaThrSerThrSerThrAlaGlyThr                              180185190                                                                     AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT631                           SerHisLeuValLysSerAlaGluLysGluLysThrPheCysValAsn                              195200205                                                                     GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC679                           GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              210215220                                                                     TTGTGCAAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAGAAT727                           LeuCysLysCysGlnProGlyPheThrGlyAlaArgCysThrGluAsn                              225230235240                                                                  GTGCCCATGAAAGTCCAAACCCAAGAAAGTGCCCAAATGAGTTTACTG775                           ValProMetLysValGlnThrGlnGluSerAlaGlnMetSerLeuLeu                              245250255                                                                     GTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCTACAGTACGTCCACTC826                        ValIleAlaAlaLysThrThr                                                         260                                                                           CCTTTCTGTCTCTGCCTGAATAGCGCATCTCAGTCGGTGCCGCTTTCTTGTTGCCGCATC886               TCCCCTCAGATTCCTCCTAGAGCTAGATGCGTTTTACCAGGTCTAACATTGACTGCCTCT946               GCCTGTCGCATGAGAACATTAACACAAGCGATTGTATGACTTCCTCTGTCCGTGACTAGT1006              GGGCTCTGAGCTACTCGTAGGTGCGTAAGGCTCCAGTGTTTCTGAAATTGATCTTGAATT1066              ACTGTGATACGACATGATAGTCCCTCTCACCCAGTGCAATGACAATAAAGGCCTTGAAAA1126              GTCAAAAAAAAAAAAAAAAAAAAAAATCGATGTCGACTCGAGATGTGGCTGCAGGTCGAC1186              TCTAGAG1193                                                                   (2) INFORMATION FOR SEQ ID NO: 135:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1108                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:                                    CCTGCAGCATCAAGTGTGGGCGGCGAAAGCCGGGGGCTTGAAGAAGGAC                             HisGlnValTrpAlaAlaLysAlaGlyGlyLeuLysLysAsp                                    1510                                                                          TCGCTG55                                                                      SerLeu                                                                        15                                                                            CTCACCGTGCGCCTGGGCGCCTGGGGCCACCCCGCCTTCCCCTCCTGC103                           LeuThrValArgLeuGlyAlaTrpGlyHisProAlaPheProSerCys                              202530                                                                        GGGCGCCTCAAGGAGGACAGCAGGTACATCTTCTTCATGGAGCCCGAG151                           GlyArgLeuLysGluAspSerArgTyrIlePhePheMetGluProGlu                              354045                                                                        GCCAACAGCAGCGGCGGGCCCGGCCGCCTTCCGAGCCTCCTTCCCCCC199                           AlaAsnSerSerGlyGlyProGlyArgLeuProSerLeuLeuProPro                              505560                                                                        TCTCGAGACGGGCCGGAACCTCAAGAAGGAGGTCAGCCGGGTGCTGTG247                           SerArgAspGlyProGluProGlnGluGlyGlyGlnProGlyAlaVal                              65707580                                                                      CAACGGTGCGCCTTGCCTCCCCGCTTGAAAGAGATGAAGAGTCAGGAG295                           GlnArgCysAlaLeuProProArgLeuLysGluMetLysSerGlnGlu                              859095                                                                        TCTGTGGCAGGTTCCAAACTAGTGCTTCGGTGCGAGACCAGTTCTGAA343                           SerValAlaGlySerLysLeuValLeuArgCysGluThrSerSerGlu                              100105110                                                                     TACTCCTCTCTCAAGTTCAAGTGGTTCAAGAATGGGAGTGAATTAAGC391                           TyrSerSerLeuLysPheLysTrpPheLysAsnGlySerGluLeuSer                              115120125                                                                     CGAAAGAACAAACCAGAAAACATCAAGATACAGAAAAGGCCGGGGAAG439                           ArgLysAsnLysProGluAsnIleLysIleGlnLysArgProProLys                              130135140                                                                     TCAGAACTTCGCATTAGCAAAGCGTCACTGGCTGATTCTGGAGAATAT487                           SerGluLeuArgIleSerLysAlaSerLeuAlaAspSerGlyGluTyr                              145150155160                                                                  ATGTGCAAAGTGATCAGCAAACTAGGAAATGACAGTGCCTCTGCCAAC535                           MetCysLysValIleSerLysLeuGlyAsnAspSerAlaSerAlaAsn                              165170175                                                                     ATCACCATTGTGGAGTCAAACGCCACATCCACATCTACAGCTGGGACA583                           IleArgIleValGluSerAsnAlaThrSerThrSerThrAlaGlyThr                              180185190                                                                     AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT631                           SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              195200205                                                                     GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC679                           GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              210215220                                                                     TTGTGCAAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTAC727                           LeuCysLysCysProAsnGluPheThrGlyAspArgCysGlnAsnTyr                              225230235240                                                                  GTAATGGCCAGCTTCTACAGTACGTCCACTCCCTTTCTGTCTCTGCCT775                           ValMetAlaSerPheTyrSerThrSerThrProPheLeuSerLeuPro                              245250255                                                                     GAATAGCGCATCTCAGTCGGTGCCGCTTTCTTGTTGCCGCATCTCCCCTCAGA                         Glu                                                                           TTCCGCCTAG838                                                                 AGCTAGATGCGTTTTACCAGGTCTAACATTGACTGCCTCTGCCTGTCGCATGAGAACATT898               AACACAAGCGATTGTATGACTTCCTCTGTCCGTGACTAGTGGGCTCTGAGCTACTCGTAG958               GTGCGTAAGGCTCCAGTGTTTCTGAAATTGATCTTGAATTACTGTGATACGACATGATAG1018              TCCCTCTCACCCAGTGCAATGACAATAAAGGCCTTGAAAAGTCAAAAAAAAAAAAAAAAA1078              AAAAATCGATGTCGACTCGAGATGTGGCTG1108                                            (2) INFORMATION FOR SEQ ID NO: 136:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 559                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N in position 214 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:                                    AGTTTCCCCCCCCAACTTGTCGGAACTCTGGGCTCGCGCGCAGGGCAGGAGCGGAGCGGC60                GGCGGCTGCCCAGGCGATGCGAGCGCGGGCCGGACGGTAATCGCCTCTCCCTCCTCGGGC120               TGCGAGCGCGCCGGACCGAGGCAGCGACAGGAGCGGACCGCGGCGGGAACCGAGGACTCC180               CCAGCGGCGCGCCAGCAGGAGCCACCCCGCGAGNCGTGCGACCGGGACGGAGCGCCCGCC240               AGTCCCAGGTGGCCCGGACCGCACGTTGCGTCCCCGCGCTCCCCGCCGGCGACAGGAGAC300               GCTCCCCCCCACGCCGCGCGCGCCTCGGCCCGGTCGCTGGCCCGCCTCCACTCCGGGGAC360               AAACTTTTCCCGAAGCCGATCCCAGCCCTCGGACCCAAACTTGTCGCGCGTCGCCTTCGC420               CGGGAGCCGTCCGCGCAGAGCGTGCACTTCTCGGGCGAGATGTCGGAGCGCAGA474                     MetSerGluArgArg                                                               15                                                                            GAAGGCAAAGGCAAGGGGAAGGGCGGCAAGAAGGACCGAGGCTCCGGG522                           GluGlyLysGlyLysGlyLysGlyGlyLysLysAspArgGlySerGly                              101520                                                                        AAGAAGCCCGTGCCCGCGGCTGGCGGCCCGAGCCCAG559                                      LysLysProValProAlaAlaGlyGlyProSerProAla                                       2530                                                                          (2) INFORMATION FOR SEQ ID NO: 137:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 252                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N in position 8 could be either A                      or G.                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:                                    CCCATCANGTGTGGGCGGCGAAAGCCGGGGGCTTGAAGAAGGACTCG47                             HisGlnValTrpAlaAlaLysAlaGlyGlyLeuLysLysAspSer                                 151015                                                                        CTGCTCACCGTGCGCCTGGGCGCCTGGGGCCACCCCGCCTTCCCCTCC95                            LeuLeuThrValArgLeuGlyAlaTrpGlyHisProAlaPheProSer                              202530                                                                        TGCGGGCGCCTCAAGGAGGACAGCAGGTACATCTTCTTCATGGAGCCC143                           CysGlyArgLeuLysGluAspSerArgTyrIlePhePheMetGluPro                              354045                                                                        GAGGCCAACAGCAGCGGCGGGCCCGGCCGCCTTCCGAGCCTCCTTCCC191                           GluAlaAsnSerSerGlyGlyProGlyArgLeuProSerLeuLeuPro                              505560                                                                        CCCTCTCGAGACGGGCCGGAACCTCAAGAAGGAGGTCAGCCGGGTGCT239                           ProSerArgAspGlyProGluProGlnGluGlyGlyGlnProGlyAla                              657075                                                                        GTGCAACGGTGCG252                                                              ValGlnArgCys                                                                  80                                                                            (2) INFORMATION FOR SEQ ID NO: 138:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 178                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:                                    CCTTGCCTCCCCGCTTGAAAGAGATGAAGAGTCAGGAGTCTGTGGCAG48                            LeuProProArgLeuLysGluHisLysSerGlnGluSerValAlaGly                              151015                                                                        GTTCCAAACTAGTGCTTCGGTGCGAGACCAGTTCTGAATACTCCTCTC96                            SerLysLeuValLeuArgCysGluThrSerSerGluTyrSerSerLeu                              202530                                                                        TCAAGTTCAAGTGGTTCAAGAATGGGAGTGAATTAAGCCGAAAGAACA144                           LysPheLysTrpPheLysAsnGlySerGluLeuSerArgLysAsnLys                              354045                                                                        AACCACAAAACATCAAGATACAGAAAAGGCCGGG178                                         ProGlyAsnIleLysIleGlnLysArgProGly                                             5055                                                                          (2) INFORMATION FOR SEQ ID NO: 139:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 122                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:                                    GAAGTCAGAACTTCGCATTAGCAAAGCGTCACTGGCTGATTCTGGA46                              LysSerGluLeuArgIleSerLysAlaSerLeuAlaAspSerGly                                 151015                                                                        GAATATATGTGCAAAGTGATCAGCAAACTAGGAAATGACAGTGCCTCT94                            GluTyrMetCysLysValIleSerLysLeuGlyAsnAspSerAlaSer                              202530                                                                        GCCAACATCACCATTGTGGAGTCAAACG122                                               AlaAsnIleThrIleValGluSerAsnAla                                                35                                                                            (2) INFORMATION FOR SEQ ID NO: 140:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140:                                    TCTAAAACTACAGAGACTGTATTTTCATGATCATCATAGTTCTGTGAAATATACTTAAAC60                CGCTTTGGTCCTGATCTTGTAGGAAGTCAGAACTTCGCATTAGCAAAGCG110                         LysSerGluLeuArgIleSerLysAla                                                   15                                                                            TCACTGGCTGATTCTGGAGAATATATGTGCAAAGTGATCAGCAAACTA158                           SerLeuAlaAspSerGlyGluTyrMetCysLysValIleSerLysLeu                              10152025                                                                      GGAAATGACAGTGCCTCTGCCAACATCACCATTGTGGAGTCAAACGGT206                           GlyAsnAspSerAlaSerAlaAsnIleThrIleValGluSerAsnGly                              303540                                                                        AAGAGATGCCTACTGCGTGCTATTTCTCAGTCTCTAAGAGGAGTGATC254                           LysArgCysLeuLeuArgAlaIleSerGlnSerLeuArgGlyValIle                              455055                                                                        AAGGTATGTGGTCACACTTGAATCACGCAGGTGTGTGAAATCTCATTG302                           LysValCysGlyHisThr                                                            60                                                                            TGAACAAATAAAAATCATGAAAGGAAAACTCTATGTTTGAAATATCTTATGGGTCCTCCT362               GTAAAGCTCTTCACTCCATAAGGTGAAATAGACCTGAAATATATATAGATTATTT417                    (2) INFORMATION FOR SEQ ID NO: 141:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:                                    AGATCACCACTGGCATGCCAGCCTCAACTGAGACAGCGTATGTGTCT47                             GluIleThrThrGlyMetProAlaSerThrGluThrAlaTyrValSer                              151015                                                                        TCAGAGTCTCCCATTAGAATATCAGTATCAACAGAAGGAACAAATACT95                            SerGluSerProIleArgIleSerValSerThrGluGlyThrAsnThr                              202530                                                                        TCTTCAT102                                                                    SerSerSer                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 142:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142:                                    AAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAGAATGTGCCC48                            LysCysGlnProGlyPheThrGlyAlaArgCysThrGluAsnValPro                              151015                                                                        ATGAAAGTCCAAACCCAAGAA69                                                       MetLysValGlnThrGlnGlu                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO: 143:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:                                    AAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTACGTAATG48                            LysCysProAsnGluPheThrGlyAspArgCysGlnAsnTyrValMet                              151015                                                                        GCCAGCTTCTAC60                                                                AlaSerPheTyr                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO: 144:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144:                                    AGTACGTCCACTCCCTTTCTGTCTCTGCCTGAATAG36                                        SerThrSerThrProPheLeuSerLeuProGlu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO: 145:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145:                                    AAGCATCTTGGGATTGAATTTATGGAG27                                                 LysHisLeuGlyIleGluPheMetGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 146:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 569                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146:                                    AAAGCGGAGGAGCTCTACCAGAAGAGAGTGCTCACCATTACCGGCATT48                            LysAlaGluGluLeuTyrGlnLysArgValLeuThrIleThrGlyIle                              151015                                                                        TGCATCGCGCTGCTCGTGGTTGGCATCATGTGTGTGGTGGTCTACTGC96                            CysIleAlaLeuLeuValValGlyIleMetCysValValValTyrCys                              202530                                                                        AAAACCAAGAAACAACGGAAAAAGCTTCATGACCGGCTTCGGCAGAGC144                           LysThrLysLysGlnArgLysLysLeuHisAspArgLeuArgGlnSer                              354045                                                                        CTTCGGTCTGAAAGAAACACCATGATGAACGTAGCCAACGGGCCCCAC192                           LeuArgSerGluArgAsnThrMetMetAsnValAlaAsnGlyProHis                              505560                                                                        CACCCCAATCCGCCCCCCGAGAACGTGCAGCTGGTGAATCAATACGTA240                           HisProAsnProProProGluAsnValGlnLeuValAsnGlnTyrVal                              65707580                                                                      TCTAAAAATGTCATCTCTAGCGAGCATATTGTTGAGAGAGAGGCGGAG288                           SerLysAsnValIleSerSerGluHisIleValGluArgGluAlaGlu                              859095                                                                        AGCTCTTTTTCCACCAGTCACTACACTTCGACAGCTCATCATTCCACT336                           SerSerPheSerThrSerHisTyrThrSerThrAlaHisHisSerThr                              100105110                                                                     ACTGTCACTCAGACTCCCAGTCACAGCTGGAGCAATGGACACACTGAA384                           ThrValThrGlnThrProSerHisSerTrpSerAsnGlyHisThrGlu                              115120125                                                                     AGCATCATTTCGGAAAGCCACTCTGTCATCGTGATGTCATCCGTAGAA432                           SerIleIleSerGluSerHisSerValIleValMetSerSerValGlu                              130135140                                                                     AACAGTAGGCACAGCAGCCCGACTGGGGGCCCGAGAGGACGTCTCAAT480                           AsnSerArgHisSerSerProThrGlyGlyProArgGlyArgLeuAsn                              145150155160                                                                  GGCTTGGGAGGCCCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGA528                           GlyLeuGlyGlyProArgGluCysAsnSerPheLeuArgHisAlaArg                              165170175                                                                     GAAACCCCTGACTCCTACCGAGACTCTCCTCATAGTGAAAG569                                  GluThrProAspSerTyrArgAspSerProHisSer                                          180185                                                                        (2) INFORMATION FOR SEQ ID NO: 147:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 730                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147:                                    GTATGTATCAGCAATGACCACCCCGGCTCGTATGTCACCTGTAGAT46                              TyrValSerAlaMetThrThrProAlaArgMetSerProValAsp                                 151015                                                                        TTCCACACGCCAAGCTCCCCCAAGTCACCCCCTTCGGAAATGTCCCCG94                            PheHisThrProSerSerProLysSerProProSerGluMetSerPro                              202530                                                                        CCCGTGTCCAGCACGACGGTCTCCATGCCCTCCATGGCGGTCAGTCCC142                           ProValSerSerThrThrValSerMetProSerMetAlaValSerPro                              354045                                                                        TTCGTGGAAGAGGAGAGACCCCTGCTCCTTGTGACGCCACCACGGCTG190                           PheValGluGluGluArgProLeuLeuLeuValThrProProArgLeu                              505560                                                                        CGGGAGAAGTATGACCACCACGCCCAGCAATTCAACTCGTTCCACTGC238                           ArgGluLysTyrAspHisHisAlaGlnGlnPheAsnSerPheHisCys                              657075                                                                        AACCCCGCGCATGAGAGCAACAGCCTGCCCCCCAGCCCCTTGAGGATA286                           AsnProAlaHisGluSerAsnSerLeuProProSerProLeuArgIle                              80859095                                                                      GTGGAGGATGAGGAATATGAAACGACCCAGGAGTACGAACCAGCTCAA334                           ValGluAspGluGluTyrGluThrThrGlnGluTyrGluProAlaGln                              100105110                                                                     GAGCCGGTTAAGAAACTCACCAACAGCAGCCGGCGGGCCAAAAGAACC382                           GluProValLysLysLeuThrAsnSerSerArgArgAlaLysArgThr                              115120125                                                                     AAGCCCAATGGTCACATTGCCCACAGGTTGGAAATGGACAACAACACA430                           LysProAsnGlyHisIleAlaHisArgLeuGluMetAspAsnAsnThr                              130135140                                                                     GGCGCTGACAGCAGTAACTCAGAGAGCGAAACAGAGGATGAAAGAGTA478                           GlyAlaAspSerSerAsnSerGluSerGluThrGluAspGluArgVal                              145150155                                                                     GGAGAAGATACGCCTTTCCTGGCCATACAGAACCCCCTGGCAGCCAGT526                           GlyGluAspThrProPheLeuAlaIleGlnAsnProLeuAlaAlaSer                              160165170175                                                                  CTCGAGGCGGCCCCTGCCTTCCGCCTGGTCGACAGCAGGACTAACCCA574                           LeuGluAlaAlaProAlaPheArgLeuValAspSerArgThrAsnPro                              180185190                                                                     ACAGGCGGCTTCTCTCCGCAGGAAGAATTGCAGGCCAGGCTCTCCGGT622                           ThrGlyGlyPheSerProGlnGluGluLeuGlnAlaArgLeuSerGly                              195200205                                                                     GTAATCGCTAACCAAGACCCTATCGCTGTCTAAAACCGAAATACACCCAT672                         ValIleAlaAsnGlnAspProIleAlaVal                                                210215                                                                        AGATTCACCTGTAAAACTTTATTTTATATAATAAAGTATTCCACCTTAAATTAAACAA730                 (2) INFORMATION FOR SEQ ID NO: 148:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1652                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148:                                    AGTTTCCCCCCCCAACTTGTCGGAACTCTGGGCTCGCGCGCAGGGCAGGAGCGGAGCGGC60                GGCGGCTGCCCAGGCGATGCGAGCGCGGGCCGGACGGTAATCGCCTCTCCCTCCTCGGGC120               TGCGAGCGCGCCGGACCGAGGCAGCGACAGGAGCGGACCGCGGCGGGAACCGAGGACTCC180               CCAGCGGCGCGCCAGCAGGAGCCACCCCGCGAGCGTGCGACCGGGACGGAGCGCCCGCCA240               GTCCCAGGTGGCCCGGACCGCACGTTGCGTCCCCGCGCTCCCCGCCGGCGACAGGAGACG300               CTCCCCCCCACGCCGCGCGCGCCTCGGCCCGGTCGCTGGCCCGCCTCCACTCCGGGGACA360               AACTTTTCCCGAAGCCGATCCCAGCCCTCGGACCCAAACTTGTCGCGCGTCGCCTTCGCC420               GGGAGCCGTCCGCGCAGAGCGTGCACTTCTCGGGCGAGATGTCGGAGCGCAGA473                      MetSerGluArgArg                                                               15                                                                            GAAGGCAAAGGCAAGGGGAAGGGCGGCAAGAAGGACCGAGGCTCCGGG521                           GluGlyLysGlyLysGlyLysGlyGlyLysLysAspArgGlySerGly                              101520                                                                        AAGAAGCCCGTGCCCGCGGCTGGCGGCCCGAGCCCAGCCTTGCCTCCC569                           LysLysProValProAlaAlaGlyGlyProSerProAlaLeuProPro                              253035                                                                        CGCTTGAAAGAGATGAAGATGCAGGAGTCTGTGGCAGGTTCCAAACTA617                           ArgLeuLysGluMetLysSerGlnGluSerValAlaGlySerLysLeu                              404550                                                                        GTGCTTCGGTGCGAGACCAGTTCTGAATACTCCTCTCTCAAGTTCAAG665                           ValLeuArgCysGluThrSerSerGluTyrSerSerLeuLysPheLys                              556065                                                                        TGGTTCAAGAATGGGAGTGAATTAAGCCGAAAGAACAAACCACAAAAC713                           TrpPheLysAsnGlySerGluLeuSerArgLysAsnLysProGlnAsn                              70758085                                                                      ATCAAGATACAGAAAAGGCCGGGGAAGTCAGAACTTCGCATTAGCAAA761                           IleLysIleGlnLysArgProGlyLysSerGluLeuArgIleSerLys                              9095100                                                                       GCGTCACTGGCTGATTCTGGAGAATATATGTGCAAAGTGATCAGCAAA809                           AlaSerLeuAlaAspSerGlyGluTyrMetCysLysValIleSerLys                              105110115                                                                     CTAGGAAATGACAGTGCCTCTGCCAACATCACCATTGTGGAGTCAAAC857                           LeuGlyAsnAspSerAlaSerAlaAsnIleThrIleValGluSerAsn                              120125130                                                                     GAGATCACCACTGGCATGCCAGCCTCAACTGAGACAGCGTATGTGTCT905                           GluIleThrThrGlyMetProAlaSerThrGluThrAlaTyrValSer                              135140145                                                                     TCAGAGTCTCCCATTAGAATATCAGTATCAACAGAAGGAACAAATACT953                           SerGluSerProIleArgIleSerValSerThrGluGlyThrAsnThr                              150155160165                                                                  TCTTCATCCACATCCACATCTACAGCTGGGACAAGCCATCTTGTCAAG1001                          SerSerSerThrSerThrSerThrAlaGlyThrSerHisLeuValLys                              170175180                                                                     TGTGCAGAGAAGGAGAAAACTTTCTGTGTGAATGGAGGCGAGTGCTTC1049                          CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPhe                              185190195                                                                     ATGGTGAAAGACCTTTCAAATCCCTCAAGATACTTGTGCAAGTGCCCA1097                          MetValLysAspLeuSerAsnProSerArgTyrLeuCysLysCysPro                              200205210                                                                     AATGAGTTTACTGGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTC1145                          AsnGluPheThrGlyAspArgCysGlnAsnTyrValMetAlaSerPhe                              215220225                                                                     TACAGTACGTCCACTCCCTTTCTGTCTCTGCCTGAATAGGCGCATG1191                            TyrSerThrSerThrProPheLeuSerLeuProGlu                                          230235240                                                                     CTCAGTCGGTGCCGCTTTCTTGTTGCCGCATCTCCCCTCAGATTCAACCTAGAGCTAGAT1251              GCGTTTTACCAGGTCTAACATTGACTGCCTCTGCCTGTCGCATGAGAACATTAACACAAG1311              CGATTGTATGACTTCCTCTGTCCGTGACTAGTGGGCTCTGAGCTACTCGTAGGTGCGTAA1371              GGCTCCAGTGTTTCTGAAATTGATCTTGAATTACTGTGATACGACATGATAGTCCCTCTC1431              ACCCAGTGCAATGACAATAAAGGCCTTGAAAAGTCTCACTTTTATTGAGAAAATAAAAAT1491              CGTTCCACGGGACAGTCCCTCTTCTTTATAAAATGACCCTATCCTTGAAAAGGAGGTGTG1551              TTAAGTTGTAACCAGTACACACTTGAAATGATGGTAAGTTCGCTTCGGTTCAGAATGTGT1611              TCTTTCTGACAAATAAACAGAATAAAAAAAAAAAAAAAAAA1652                                 (2) INFORMATION FOR SEQ ID NO: 149:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1140                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149:                                    CATCANGTGTGGGCGGCGAAAGCCGGGGGCTTGAAGAAGGACTCGCTG48                            HisGlnValTrpAlaAlaLysAlaGlyGlyLeuLysLysAspSerLeu                              151015                                                                        CTCACCGTGCGCCTGGGCGCCTGGGGCCACCCCGCCTTCCCCTCCTGC96                            LeuThrValArgLeuGlyAlaTrpGlyHisProAlaPheProSerCys                              202530                                                                        GGGCGCCTCAAGGAGGACAGCAGGTACATCTTCTTCATGGAGCCCGAG144                           GlyArgLeuLysGluAspSerArgTyrIlePhePheMetGluProGlu                              354045                                                                        GCCAACAGCAGCGGCGGGCCCGGCCGCCTTCCGAGCCTCCTTCCCCCC192                           AlaAsnSerSerGlyGlyProGlyArgLeuProSerLeuLeuProPro                              505560                                                                        TCTCGAGACGGGCCGGAACCTCAAGAAGGAGGTCAGCCGGGTGCTGTG240                           SerArgAspGlyProGluProGlnGluGlyGlyGlnProGlyAlaVal                              65707580                                                                      CAACGGTGCGCCTTGCCTCCCCGCTTGAAAGAGATGAAGAGTCAGGAG288                           GlnArgCysAlaLeuProProArgLeuLysGluMetLysSerGlnGlu                              859095                                                                        TCTGTGGCAGGTTCCAAACTAGTGCTTCGGTGCGAGACCAGTTCTGAA336                           SerValAlaGlySerLysLeuValLeuArgCysGluThrSerSerGlu                              100105110                                                                     TACTCCTCTCTCAAGTTCAAGTGGTTCAAGAATGGGAGTGAATTAAGC384                           TyrSerSerLeuLysPheLysTrpPheLysAsnGlySerGluLeuSer                              115120125                                                                     CGAAAGAACAAACCAGAAAACATCAAGATACAGAAAAGGCCGGGGAAG432                           ArgLysAsnLysProGluAsnIleLysIleGlnLysArgProGlyLys                              130135140                                                                     TCAGAACTTCGCATTAGCAAAGCGTCACTGGCTGATTCTGGAGAATAT480                           SerGluLeuArgIleSerLysAlaSerLeuAlaAspSerGlyGluTyr                              145150155160                                                                  ATGTGCAAAGTGATCAGCAAACTAGGAAATGACAGTGCCTCTGCCAAC528                           MetCysLysValIleSerLysLeuGlyAsnAspSerAlaSerAlaAsn                              165170175                                                                     ATCACCATTGTGGAGTCAAACGCCACATCCACATCTACAGCTGGGACA576                           IleThrIleValGluSerAsnAlaThrSerThrSerThrAlaGlyThr                              180185190                                                                     AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT624                           SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              195200205                                                                     GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC672                           GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              210215220                                                                     TTGTGCAAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAGAAT720                           LeuCysLysCysGlnProGlyPheThrGlyAlaArgCysThrGluAsn                              225230235240                                                                  GTGCCCATGAAAGTCCAAACCCAAGAAAAGTGCCCAAATGAGTTTACT768                           ValProMetLysValGlnThrGlnGluLysCysProAsnGluPheThr                              245250255                                                                     GGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCTACAGTACGTCC816                           GlyAspArgCysGlnAsnTyrValMetAlaSerPheTyrSerThrSer                              260265270                                                                     ACTCCCTTTCTGTCTCTGCCTGAATAGCGCATCTCAGTCGGTGCCGCTTTCTTG870                     ThrProPheLeuSerLeuProGlu                                                      275280                                                                        TTGCCGCATCTCCCCTCAGATTCCNCCTAGAGCTAGATGCGTTTTACCAGGTCTAACATT930               GACTGCCTCTGCCTGTCGCATGAGAACATTAACACAAGCGATTGTATGACTTCCTCTGTC990               CGTGACTAGTGGGCTCTGAGCTACTCGTAGGTGCGTAAGGCTCCAGTGTTTCTGAAATTG1050              ATCTTGAATTACTGTGATACGACATGATAGTCCCTCTCACCCAGTGCAATGACAATAAAG1110              GCCTTGAAAAGTCAAAAAAAAAAAAAAAAA1140                                            (2) INFORMATION FOR SEQ ID NO: 150:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1764                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:                                    GAAGTCAGAACTTCGCATTAGCAAAGCGTCACTGGCTGATTCTGGAGAA49                           LysSerGluLeuArgIleSerLysAlaSerLeuAlaAspSerGlyGlu                              151015                                                                        TATATGTGCAAAGTGATCAGCAAACTAGGAAATGACAGTGCCTCTGCC97                            TyrMetCysLysValIleSerLysLeuGlyAsnAspSerAlaSerAla                              202530                                                                        AACATCACCATTGTGGAGTCAAACGCCACATCCACATCTACAGCTGGG145                           AsnIleThrIleValGluSerAsnAlaThrSerThrSerThrAlaGly                              354045                                                                        ACAAGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTG193                           ThrSerHisLeuValLysCysAlaGluLysGluLysThrPheCysVal                              505560                                                                        AATGGAGGCGACTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGA241                           AsnGlyGlyAspCysPheMetValLysAspLeuSerAsnProSerArg                              65707580                                                                      TACTTGTGCAAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAG289                           TyrLeuCysLysCysGlnProGlyPheThrGlyAlaArgCysThrGlu                              859095                                                                        AATGTGCCCATGAAAGTCCAAACCCAAGAAAAAGCGGAGGAGCTCTAC337                           AsnValProMetLysValGlnThrGlnGluLysAlaGluGluLeuTyr                              100105110                                                                     CAGAAGAGAGTGCTCACCATTACCGGCATTTGCATCGCGCTGCTCGTG385                           GlnLysArgValLeuThrIleThrGlyIleCysIleAlaLeuLeuVal                              115120125                                                                     GTTGGCATCATGTGTGTGGTGGTCTACTGCAAAACCAAGAAACAACGG433                           ValGlyIleMetCysValValValTyrCysLysThrLysLysGlnArg                              130135140                                                                     AAAAAGCTTCATGACCGGCTTCGGCAGAGCCTTCGGTCTGAAAGAAAC481                           LysLysLeuHisAspArgLeuArgGlnSerLeuArgSerGluArgAsn                              145150155160                                                                  ACCATGATGAACGTAGCCAACGGGCCCCACCACCCCAATCCGCCCCCC529                           ThrMetMetAsnValAlaAsnGlyProHisHisProAsnProProPro                              165170175                                                                     GAGAACGTGCAGCTGGTGAATCAATACGTATCTAAAAATGTCATCTCT577                           GluAsnValGlnLeuValAsnGlnTyrValSerLysAsnValIleSer                              180185190                                                                     AGCGAGCATATTGTTGAGAGAGAGGCGGAGAGCTCTTTTTCCACCAGT625                           SerGluHisIleValGluArgGluAlaGluSerSerPheSerThrSer                              195200205                                                                     CACTACACTTCGACAGCTCATCATTCCACTACTGTCACTCAGACTCCC673                           HisTyrThrSerThrAlaHisHisSerThrThrValThrGlnThrPro                              210215220                                                                     AGTCACAGCTGGAGCAATGGACACACTGAAAGCATCATTTCGGAAAGC721                           SerHisSerTrpSerAsnGlyHisThrGluSerIleIleSerGluSer                              225230235240                                                                  CACTCTGTCATCGTGATGTCATCCGTAGAAAACAGTAGGCACAGCAGC769                           HisSerValIleValMetSerSerValGluAsnSerArgHisSerSer                              245250255                                                                     CCGACTGGGGGCCCGAGAGGACGTCTCAATGGCTTGGGAGGCCCTCGT817                           ProThrGlyGlyProArgGlyArgLeuAsnGlyLeuGlyGlyProArg                              260265270                                                                     GAATGTAACAGCTTCCTCAGGCATGCCAGAGAAACCCCTGACTCCTAC865                           GluCysAsnSerPheLeuArgHisAlaArgGluThrProAspSerTyr                              275280285                                                                     CGAGACTCTCCTCATAGTGAAAGACATAACCTTATAGCTGAGCTAAGG913                           ArgAspSerProHisSerGluArgHisAsnLeuIleAlaGluLeuArg                              290295300                                                                     AGAAACAAGGCCCACAGATCCAAATGCATGCAGATCCAGCTTTCCGCA961                           ArgAsnLysAlaHisArgSerLysCysMetGlnIleGlnLeuSerAla                              305310315320                                                                  ACTCATCTTAGAGCTTCTTCCATTCCCCATTGGGCTTCATTCTCTAAG1009                          ThrHisLeuArgAlaSerSerIleProHisTrpAlaSerPheSerLys                              325330335                                                                     ACCCCTTGGCCTTTAGGAAGGTATGTATCAGCAATGACCACCCCGGCT1057                          ThrProTrpProLeuGlyArgTyrValSerAlaMetThrThrProAla                              340345350                                                                     CGTATGTCACCTGTAGATTTCCACACGCCAAGCTCCCCCAAGTCACCC1105                          ArgMetSerProValAspPheHisThrProSerSerProLysSerPro                              355360365                                                                     CCTTCGGAAATGTCCCCGCCCGTGTCCAGCACGACGGTCTCCATGCCC1153                          ProSerGluMetSerProProValSerSerThrThrValSerMetPro                              370375380                                                                     TCCATGGCGGTCAGTCCCTTCGTGGAAGAGGAGAGACCCCTGCTCCTT1201                          SerMetAlaValSerProPheValGluGluGluArgProLeuLeuLeu                              385390395400                                                                  GTGACGCCACCACGGCTGCGGGAGAAGTATGACCACCACGCCCAGCAA1249                          ValThrProProArgLeuArgGluLysTyrAspHisHisAlaGlnGln                              405410415                                                                     TTCAACTCGTTCCACTGCAACCCCGCGCATGAGAGCAACAGCCTGCCC1297                          PheAsnSerPheHisCysAsnProAlaHisGluSerAsnSerLeuPro                              420425430                                                                     CCCAGCCCCTTGAGGATAGTGGAGGATGAGGAATATGAAACGACCCAG1345                          ProSerProLeuArgIleValGluAspGluGluTyrGluThrThrGln                              435440445                                                                     GAGTACGAACCAGCTCAAGAGCCGGTTAAGAAACTCACCAACAGCAGC1393                          GluTyrGluProAlaGlnGluProValLysLysLeuThrAsnSerSer                              450455460                                                                     CGGCGGGCCAAAAGAACCAAGCCCAATGGTCACATTGCCCACAGGTTG1441                          ArgArgAlaLysArgThrLysProAsnGlyHisIleAlaHisArgLeu                              465470475480                                                                  GAAATGGACAACAACACAGGCGCTGACAGCAGTAACTCAGAGAGCGAA1489                          GluMetAspAsnAsnThrGlyAlaAspSerSerAsnSerGluSerGlu                              485490495                                                                     ACAGAGGATGAAAGAGTAGGAGAAGATACGCCTTTCCTGGCCATACAG1537                          ThrGluAspGluArgValGlyGluAspThrProPheLeuAlaIleGln                              500505510                                                                     AACCCCCTGGCAGCCAGTCTCGAGGCGGCCCCTGCCTTCCGCCTGGTC1585                          AsnProLeuAlaAlaSerLeuGluAlaAlaProAlaPheArgLeuVal                              515520525                                                                     GACAGCAGGACTAACCCAACAGGCGGCTTCTCTCCGCAGGAAGAATTG1633                          AspSerArgThrAsnProThrGlyGlyPheSerProGlnGluGluLeu                              530535540                                                                     CAGGCCAGGCTCTCCGGTGTAATCGCTAACCAAGACCCTATCGCTGTC1681                          GlnAlaArgLeuSerGlyValIleAlaAsnGlnAspProIleAlaVal                              545550555560                                                                  TAAAACCGAAATACACCCATAGATTCACCTGTAAAACTTTATTTTATATAATAAAGTATT1741              CCACCTTAAATTAAACAAAAAAA1764                                                   (2) INFORMATION FOR SEQ ID NO: 151:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151:                                    LysCysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCys                              151015                                                                        PheMetValLysAspLeuSerAsnProSerArgTyrLeuCysLysCys                              202530                                                                        ProAsnGluPheThrGlyAspArgCysGlnAsnTyrValMetAlaSer                              354045                                                                        PheTyr                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO: 152:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152:                                    LysCysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCys                              151015                                                                        PheMetValLysAspLeuSerAsnProSerArgTyrLeuCysLysCys                              202530                                                                        GlnProGlyPheThrGlyAlaArgCysThrGluAsnValProMetLys                              354045                                                                        ValGln                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO: 153:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153:                                    GluCysLeuArgLysTyrLysAspPheCysIleHisGlyGluCysLys                              151015                                                                        TyrValLysGluLeuArgAlaProSerCysLysCysGlnGlnGluTyr                              202530                                                                        PheGlyGluArgCysGlyGluLysSerAsnLysThrHisSer                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO: 154:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 198                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154:                                    AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT48                            SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              151015                                                                        GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC96                            GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              202530                                                                        TTGTGCAAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTAC144                           LeuCysLysCysProAsnGluPheThrGlyAspArgCysGlnAsnTyr                              354045                                                                        GTAATGGCCAGCTTCTACAGTACGTCCACTCCCTTTCTGTCTCTGCCT192                           ValMetAlaSerPheTyrSerThrSerThrProPheLeuSerLeuPro                              505560                                                                        GAATAG198                                                                     Glu                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO: 155:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 192                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155:                                    AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT48                            SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              151015                                                                        GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC96                            GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              202530                                                                        TTGTGCAAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAGAAT144                           LeuCysLysCysGlnProGlyPheThrGlyAlaArgCysThrGluAsn                              354045                                                                        GTGCCCATGAAAGTCCAAACCCAAGAAAAAGCGGAGGAGCTCTACTAA192                           ValProMetLysValGlnThrGlnGluLysAlaGluGluLeuTyr                                 505560                                                                        (2) INFORMATION FOR SEQ ID NO: 156:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 183                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156:                                    AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT48                            SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              151015                                                                        GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC96                            GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              202530                                                                        TTGTGCAAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTAC144                           LeuCysLysCysProAsnGluPheThrGlyAspArgCysGlnAsnTyr                              354045                                                                        GTAATGGCCAGCTTCTACAAAGCGGAGGAGCTCTACTAA183                                    ValMetAlaSerPheTyrLysAlaGluGluLeuTyr                                          505560                                                                        (2) INFORMATION FOR SEQ ID NO: 157:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 210                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157:                                    AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT48                            SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              151015                                                                        GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC96                            GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              202530                                                                        TTGTGCAAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTAC144                           LeuCysLysCysProAsnGluPheThrGlyAspArgCysGlnAsnTyr                              354045                                                                        GTAATGGCCAGCTTCTACAAGCATCTTGGGATTGAATTTATGGAGAAA192                           ValMetAlaSerPheTyrLysHisLeuGlyIleGluPheMetGluLys                              505560                                                                        GCGGAGGAGCTCTACTAA210                                                         AlaGluGluLeuTyr                                                               65                                                                            (2) INFORMATION FOR SEQ ID NO: 158:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 267                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158:                                    AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT48                            SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              151015                                                                        GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC96                            GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              202530                                                                        TTGTGCAAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAGAAT144                           LeuCysLysCysGlnProGlyPheThrGlyAlaArgCysThrGluAsn                              354045                                                                        GTGCCCATGAAAGTCCAAACCCAAGAAAAGTGCCCAAATGAGTTTACT192                           ValProMetLysValGlnThrGlnGluLysCysProAsnGluPheThr                              505560                                                                        GGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCTACAGTACGTCC240                           GlyAspArgCysGlnAsnTyrValMetAlaSerPheTyrSerThrSer                              65707580                                                                      ACTCCCTTTCTGTCTCTGCCTGAATAG267                                                ThrProPheLeuSerLeuProGlu                                                      85                                                                            (2) INFORMATION FOR SEQ ID NO: 159:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 252                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159:                                    AGCCATCTTGTCAAGTGTGCAGAGAAGGAGAAAACTTTCTGTGTGAAT48                            SerHisLeuValLysCysAlaGluLysGluLysThrPheCysValAsn                              151015                                                                        GGAGGCGAGTGCTTCATGGTGAAAGACCTTTCAAATCCCTCAAGATAC96                            GlyGlyGluCysPheMetValLysAspLeuSerAsnProSerArgTyr                              202530                                                                        TTGTGCAAGTGCCAACCTGGATTCACTGGAGCGAGATGTACTGAGAAT144                           LeuCysLysCysGlnProGlyPheThrGlyAlaArgCysThrGluAsn                              354045                                                                        GTGCCCATGAAAGTCCAAACCCAAGAAAAGTGCCCAAATGAGTTTACT192                           ValProMetLysValGlnThrGlnGluLysCysProAsnGluPheThr                              505560                                                                        GGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCTACAAAGCGGAG240                           GlyAspArgCysGlnAsnTyrValMetAlaSerPheTyrLysAlaGlu                              65707580                                                                      GAGCTCTACTAA252                                                               GluLeuTyr                                                                     (2) INFORMATION FOR SEQ ID NO: 160:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 128                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160:                                    CCACATCCACATCTACAGCTGGGACAAGCCATCTTGTCAAGTGTGCA47                             ThrSerThrSerThrAlaGlyThrSerHisLeuValLysCysAla                                 151015                                                                        GAGAAGGAGAAAACTTTCTGTGTGAATGGAGGCGAGTGCTTCATGGTG95                            GluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal                              202530                                                                        AAAGACCTTTCAAATCCCTCAAGATACTTGTGC128                                          LysAspLeuSerAsnProSerArgTyrLeu                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 161:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:                                    ACATAACCTTATAGCTGAGCTAAGGAGAAACAAGGCCCACAGATCC46                              HisAsnLeuIleAlaGluLeuArgArgAsnLysAlaHisArgSer                                 151015                                                                        AAATGCATGCAGATCCAGCTTTCCGCAACTCATCTTAGAGCTTCTTCC94                            LysCysMetGlnIleGlnLeuSerAlaThrHisLeuArgAlaSerSer                              202530                                                                        ATTCCCCATTGGGCTTCATTCTCTAAGACCCCTTGGCCTTTAGGAAG141                            IleProHisTrpAlaSerPheSerLysThrProTrpProLeuGlyArg                              354045                                                                        (2) INFORMATION FOR SEQ ID NO: 162:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in positions 15 and 22 is unknown.                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162:                                    AlaAlaGluLysGluLysThrPheCysValAsnGlyGlyGluXaaPhe                              151015                                                                        MetValLysAspLeuXaaAsnPro                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO: 163:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 745                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163:                                    ATGAGATGGCGACGCGCCCCGCGCCGCTCCGGGCGTCCCGGCCCCCGG48                            MetArgTrpArgArgAlaProArgArgSerGlyArgProGlyProArg                              151015                                                                        GCCCAGCGCCCCGGCTCCGCCGCCCGCTCGTCGCCGCCGCTGCCGCTG96                            AlaGlnArgProGlySerAlaAlaArgSerSerProProLeuProLeu                              202530                                                                        CTGCCACTACTGCTGCTGCTGGGGACCGCGGCCCTGGCGCCGGGGGCG144                           LeuProLeuLeuLeuLeuLeuGlyThrAlaAlaLeuAlaProGlyAla                              354045                                                                        GCGGCCGGCAACGAGGCGGCTCCCGCGGGGGCCTCGGTGTGCTACTCG192                           AlaAlaGlyAsnGluAlaAlaProAlaGlyAlaSerValCysTyrSer                              505560                                                                        TCCCCGCCCAGCGTGGGATCGGTGCAGGAGCTAGCTCAGCGCGCCGCG240                           SerProProSerValGlySerValGlnGluLeuAlaGlnArgAlaAla                              65707580                                                                      GTGGTGATCGAGGGAAAGGTGCACCCGCAGCGGCGGCAGCAGGGGGCA288                           ValValIleGluGlyLysValHisProGlnArgArgGlnGlnGlyAla                              859095                                                                        CTCGACAGGAAGGCGGCGGCGGCGGCGGGCGAGGCAGGGGCGTGGGGC336                           LeuAspArgLysAlaAlaAlaAlaAlaGlyGluAlaGlyAlaTrpGly                              100105110                                                                     GGCGATCGCGAGCCGCCAGCCGCGGGCCCACGGGCGCTGGGGCCGCCC384                           GlyAspArgGluProProAlaAlaGlyProArgAlaLeuGlyProPro                              115120125                                                                     GCCGAGGAGCCGCTGCTCGCCGCCAACGGGACCGTGCCCTCTTGGCCC432                           AlaGluGluProLeuLeuAlaAlaAsnGlyThrValProSerTrpPro                              130135140                                                                     ACCGCCCCGGTGCCCAGCGCCGGCGAGCCCGGGGAGGAGGCGCCCTAT480                           ThrAlaProValProSerAlaGlyGluProGlyGluGluAlaProTyr                              145150155160                                                                  CTGGTGAAGGTGCACCAGGTGTGGGCGGTGAAAGCCGGGGGCTTGAAG528                           LeuValLysValHisGlnValTrpAlaValLysAlaGlyGlyLeuLys                              165170175                                                                     AAGGACTCGCTGCTCACCGTGCGCCTGGGGACCTGGGGCCACCCCGCC576                           LysAspSerLeuLeuThrValArgLeuGlyThrTrpGlyHisProAla                              180185190                                                                     TTCCCCTCCTGCGGGAGGCTCAAGGAGGACAGCAGGTACATCTTCTTC624                           PheProSerCysGlyArgLeuLysGluAspSerArgTyrIlePhePhe                              195200205                                                                     ATGGAGCCCGACGCCAACAGCACCAGCCGCGCGCCGGCCGCCTTCCGA672                           MetGluProAspAlaAsnSerThrSerArgAlaProAlaAlaPheArg                              210215220                                                                     GCCTCTTTCCCCCCTCTGGAGACGGGCCGGAACCTCAAGAAGGAGGTC720                           AlaSerPheProProLeuGluThrGlyArgAsnLeuLysLysGluVal                              225230235240                                                                  AGCCGGGTGCTGTGCAAGCGGTGCG745                                                  SerArgValLeuCysLysArgCys                                                      245                                                                           (2) INFORMATION FOR SEQ ID NO: 164:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164:                                    XaaAlaLeuAlaAlaAlaGlyTyrAspValGluLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 165:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is unknown.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:                                    XaaLeuValLeuArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 166:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in positions 1, 2, and 3 is                        unknown.                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166:                                    XaaXaaXaaTyrProGlyGlnIleThrSerAsn                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO: 167:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N in positions 25 and 36 is unknown.                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167:                                    ATAGGGAAGGGCGGGGGAAGGGTCNCCCTCNGCAGGGCCGGGCTTGCCTCTGGAGCCTCT60                (2) INFORMATION FOR SEQ ID NO: 168:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N in position 16 is unknown.                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168:                                    TTTACACATATATTCNCC18                                                          (2) INFORMATION FOR SEQ ID NO: 169:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169:                                    GluThrGlnProAspProGlyGlnIleLeuLysLysValProMetVal                              151015                                                                        IleGlyAlaTyrThr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO: 170:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422                                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170:                                    MetArgTrpArgArgAlaProArgArgSerGlyArgProGlyProArg                              151015                                                                        AlaGlnArgProGlySerAlaAlaArgSerSerProProLeuProLeu                              202530                                                                        LeuProLeuLeuLeuLeuLeuGlyThrAlaAlaLeuAlaProGlyAla                              354045                                                                        AlaAlaGlyAsnGluAlaAlaProAlaGlyAlaSerValCysTyrSer                              505560                                                                        SerProProSerValGlySerValGlnGluLeuAlaGlnArgAlaAla                              65707580                                                                      ValValIleGluGlyLysValHisProGlnArgArgGlnGlnGlyAla                              859095                                                                        LeuAspArgLysAlaAlaAlaAlaAlaGlyGluAlaGlyAlaTrpGly                              100105110                                                                     GlyAspArgGluProProAlaAlaGlyProArgAlaLeuGlyProPro                              115120125                                                                     AlaGluGluProLeuLeuAlaAlaAsnGlyThrValProSerTrpPro                              130135140                                                                     ThrAlaProValProSerAlaGlyGluProGlyGluGluAlaProTyr                              145150155160                                                                  LeuValLysValHisGlnValTrpAlaValLysAlaGlyGlyLeuLys                              165170175                                                                     LysAspSerLeuLeuThrValArgLeuGlyThrTrpGlyHisProAla                              180185190                                                                     PheProSerCysGlyArgLeuLysGluAspSerArgTyrIlePhePhe                              195200205                                                                     MetGluProAspAlaAsnSerThrSerArgAlaProAlaAlaPheArg                              210215220                                                                     AlaSerPheProProLeuGluThrGlyArgAsnLeuLysLysGluVal                              225230235240                                                                  SerArgValLeuCysLysArgCysAlaLeuProProGlnLeuLysGlu                              245250255                                                                     MetLysSerGlnGluSerAlaAlaGlySerLysLeuValLeuArgCys                              260265270                                                                     GluThrSerSerGluTyrSerSerLeuArgPheLysTrpPheLysAsn                              275280285                                                                     GlyAsnGluLeuAsnArgLysAsnLysProGlnAsnIleLysIleGln                              290295300                                                                     LysLysProGlyLysSerGluLeuArgIleAsnLysAlaSerLeuAla                              305310315320                                                                  AspSerGlyGluTyrMetCysLysValIleSerLysLeuGlyAsnAsp                              325330335                                                                     SerAlaSerAlaAsnIleThrIleValGluSerAsnAlaThrSerThr                              340345350                                                                     SerThrThrGlyThrSerHisLeuValLysCysAlaGluLysGluLys                              355360365                                                                     ThrPheCysValAsnGlyGlyGluCysPheMetValLysAspLeuSer                              370375380                                                                     AsnProSerArgTyrLeuCysLysCysProAsnGluPheThrGlyAsp                              385390395400                                                                  ArgCysGlnAsnTyrValMetAlaSerPheTyrSerThrSerThrPro                              405410415                                                                     PheLeuSerLeuProGlu                                                            420                                                                           (2) INFORMATION FOR SEQ ID NO: 171:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171:                                    MetSerGluArgLysGluGlyArgGlyLysGlyLysGlyLysLysLys                              151015                                                                        GluArgGlySerGlyLysLysProGluSerAlaAlaGlySerGlnSer                              202530                                                                        ProArgGluIleIleThrGlyMetProAlaSerThrGluGlyAlaTyr                              354045                                                                        ValSerSerGluSerProIleArgIleSerValSerThrGluGlyAla                              505560                                                                        AsnThrSerSerSer                                                               65                                                                            (2) INFORMATION FOR SEQ ID NO: 172:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172:                                    ArgLysGlyAspValProGlyProArgValLysSerSerArgSerThr                              151015                                                                        ThrThrAla                                                                     (2) INFORMATION FOR SEQ ID NO: 173:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 231                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173:                                    CGCGAGCGCCTCAGCGCGGCCGCTCGCTCTCCCCCTCGAGGGACAAACTTTTCCCAAACC60                CGATCCGAGCCCTTGGACCAAACTCGCCTGCGCCGAGAGCCGTCCGCGTAGAGCGCTCCG120               TCTCCGGCGAGATGTCCGAGCGCAAAGAAGGCAGAGGCAAAGGGAAGGGCAAGAAGAAGG180               AGCGAGGCTCCGGCAAGAAGCCGGAGTCCGCGGCGGGCAGCCAGAGCCCAG231                        (2) INFORMATION FOR SEQ ID NO: 174:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 178                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174:                                    CCTTGCCTCCCCGATTGAAAGAGATGAAAAGCCAGGAATCGGCTGCAGGTTCCAAACTAG60                TCCTTCGGTGTGAAACCAGTTCTGAATACTCCTCTCTCAGATTCAAGTGGTTCAAGAATG120               GGAATGAATTGAATCGAAAAAACAAACCACAAAATATCAAGATACAAAAAAAGCCAGG178                 (2) INFORMATION FOR SEQ ID NO: 175:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 122                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175:                                    GAAGTCAGAACTTCGCATTAACAAAGCATCACTGGCTGATTCTGGAGAGTATATGTGCAA60                AGTGATCAGCAAATTAGGAAATGACAGTGCCTCTGCCAATATCACCATCGTGGAATCAAA120               CG122                                                                         (2) INFORMATION FOR SEQ ID NO: 176:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 176:                                    AGATCATCACTGGTATGCCAGCCTCAACTGAAGGAGCATATGTGTCTTCAGAGTCTCCCA60                TTAGAATATCAGTATCCACAGAAGGAGCAAATACTTCTTCAT102                                 (2) INFORMATION FOR SEQ ID NO: 177:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 128                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177:                                    CTACATCTACATCCACCACTGGGACAAGCCATCTTGTAAAATGTGCGGAGAAGGAGAAAA60                CTTTCTGTGTGAATGGAGGGGAGTGCTTCATGGTGAAAGACCTTTCAAACCCCTCGAGAT120               ACTTGTGC128                                                                   (2) INFORMATION FOR SEQ ID NO: 178:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 178:                                    AAGTGCCAACCTGGATTCACTGGAGCAAGATGTACTGAGAATGTGCCCATGAAAGTCCAA60                AACCAAGAA69                                                                   (2) INFORMATION FOR SEQ ID NO: 179:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 179:                                    TCGGGCTCCATGAAGAAGATGTA23                                                     (2) INFORMATION FOR SEQ ID NO:180:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 180:                                    TCCATGAAGAAGATGTACCTGCT23                                                     (2) INFORMATION FOR SEQ ID NO:181:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 181:                                    ATGTACCTGCTGTCCTCCTTGA22                                                      (2) INFORMATION FOR SEQ ID NO:182:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182:                                    TTGAAGAAGGACTCGCTGCTCA22                                                      (2) INFORMATION FOR SEQ ID NO:183:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 183:                                    AAAGCCGGGGGCTTGAAGAA20                                                        (2) INFORMATION FOR SEQ ID NO:184:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 184:                                    ATGARGTGTGGGCGGCGAAA20                                                        __________________________________________________________________________

What is claimed is:
 1. A basic polypeptide factor having a molecularweight, under non-reducing conditions, of from about 55 kD to about 63kD on SDS-polyacrylamide gel electrophoresis, said polypeptide factorhaving mitogenic activity stimulating the division of rat Schwann cellsin the presence of fetal calf plasma, and when isolated usingreversed-phase HPLC retains at least about 50% of said activity after 4days incubation in 0.1% trifluoroacetic acid at 4° C.
 2. Isolatedprotein encoded by SEQ ID NO:
 21. 3. Isolated protein consisting of theamino acid sequence set forth in SEQ ID NO:
 170. 4. A basic polypeptidefactor having a molecular weight under non-reducing conditions of fromabout 55 kD to about 63 kD on SDS polyacrylamide gel electrophoresis,said polypeptide factor having mitogenic activity stimulating thedivision of rat Schwann cells in the presence of fetal calf plasma.